Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA
* Author for correspondence (e-mail: jane.johnson{at}utsouthwestern.edu)
Accepted 25 November 2003
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SUMMARY |
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Key words: bHLH transcription factors, Spinal cord development, MyoD, Ngn1, Dorsal interneuron specification, Lim homeodomain factors, Neurogenesis
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Introduction |
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The neural bHLH factors belong to a much larger family of transcription
factors that are essential for diverse processes such as myogenesis
(Davis et al., 1987),
hematopoiesis (Bain et al.,
1994
) and pancreas development
(Naya et al., 1997
). The
greater HLH transcription factor family has been grouped into classes based on
tissue distribution, dimerization capabilities and DNA-binding specificities
(Massari and Murre, 2000
).
Mash1 and Math1 belong to Class II, as do other tissue-specific bHLH factors
such as the myogenic factor MyoD. Class II bHLH proteins preferentially form
heterodimers with the broadly expressed Class I bHLH factors called E-proteins
(E2a, HEB and E2-2) (Massari and Murre,
2000
). These heterodimers characteristically bind E-box DNA
consensus sites (CANNTG) and modulate transcription
(Murre et al., 1989
). Crystal
structures of MyoD (Ma et al.,
1994
) and E47 (Ellenberger et
al., 1994
) show that the HLH domain is involved in dimer
formation, and the basic region interacts with DNA. While it is probable that
in vivo these neural bHLH factors have distinct DNA targets relative to each
other and relative to other non-neural members of the family such as MyoD, in
vitro these proteins can bind similar E-box sites
(Johnson et al., 1992
) (P.
Ebert and J.E.J., unpublished data). The increase in specificity in the
function of these proteins in vivo is hypothesized to reflect additional
context-dependent protein-protein interactions or post-translational
modifications.
As predicted from the crystal structures, much of the phenotypic
specificity of the bHLH transcription factors resides within the bHLH domain
itself. Studies with the Drosophila proneural genes atonal
and scute demonstrated that the ability to promote ectopic
chordotonal organ and external sensory organ formation, respectively, resides
within the bHLH domain, with specificity arising from the basic region and the
HLH serving a required but more general function
(Chien et al., 1996).
Importantly, the residues in the basic region that interact with DNA are
common between Atonal and Scute, and amino acids that differ face away from
the DNA. Thus, it was proposed by Chien et al., that additional proteins
probably interact with the basic region to provide the specificity of activity
(Chien et al., 1996
). In
addition, studies with the muscle-specific bHLH factor MyoD, indicated that
specific residues in the basic region are important in promoting non-muscle
cells to differentiate into muscle cells
(Weintraub et al., 1991
). In
contrast, in Xenopus the domain responsible for differences in the
ability of Xash1 and X-ngnr1 (Xenopus homologs of Mash1 and Ngn) to
induce specific targets in neural development mapped to helix 1 of the HLH
domain (Talikka et al., 2002
).
Together these data demonstrate the importance of the bHLH domain to the
function of Class II bHLH transcription factors but suggest that different
domains within the bHLH are important in different cellular contexts.
For the studies reported here, we have exploited an overexpression paradigm, in ovo chick electroporation, to define distinct activities of the neural bHLH factors Mash1 and Math1. We show that each of these factors induces neuronal differentiation as defined by cell cycle exit, translocation of the cells laterally out of the ventricular zone, and expression of neuronal markers. However, overexpression of each neural bHLH factor directs distinct changes to the composition of the dorsal interneuron sub-types formed. The domains responsible for the different activities map not to the DNA interaction domains but to the HLH protein-protein interaction domains. The importance of the HLH domain for the specificity of function of each bHLH factor, rather than the DNA interaction domain, suggests that context-dependent protein-protein interactions are important for the distinct function of each neural bHLH factor.
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Materials and methods |
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Immunofluorescence
Immunofluorescence was performed by overnight incubation with the
appropriate dilution of primary antibody in PBS/1% goat serum/0.1% Triton
X-100 at 4°C, followed by hybridization with appropriate secondary
goat-anti-rabbit or goat-anti-mouse IgG, conjugated to Alexa Fluor 488 or
Alexa Fluor 594 (Molecular Probes, Inc.). Primary antibodies used for this
study include: mouse anti-Mash1 (Lo et
al., 1991), rabbit anti-LH2A/B (Lhx2/9)
(Liem et al., 1995
), mouse
anti-BOSS1 (Krämer et al.,
1991
), mouse anti-BrdU (Becton-Dickinson, Inc), mouse anti-c-Myc
(9E10 Santa Cruz Biotechnology), rabbit anti-Pax2 (Zymed), Tuj1
(Lee et al., 1990
), and from
the Developmental Studies Hybridoma Bank, 39.4D5 mouse anti-Islet1 and 4F2
mouse anti-Lim1/2 (Lhx1/5). In experiments where anti-BrdU was used, HH22-23
chick embryos were injected with 50 µl of 100 mM BrdU solution 1 hour prior
to sacrifice. Sections from BrdU-injected embryos were treated with 2 N HCL
for 15 minutes to denature the DNA, followed by a 15-minute neutralization
step with 0.1 M sodium borate pH 8.5, prior to the addition of primary
antibody. Imaging of the immunofluorescence was performed by confocal analysis
on a Bio-Rad MR-1024 confocal microscope. GFP signal was imaged using the
standard FITC filter.
Plasmid description
All electroporations utilized the expression vector pMiwIII, which drives
expression through a chick ß-actin promoter
(Muramatsu et al., 1997). The
coding regions of rat Mash1 and mouse MyoD were cloned into
the pMiwIII expression vector using convenient restriction sites. With the
exception of rat Mash1, all genes inserted into the pMiwIII vector
were myc-tagged (5 copies) at the N terminus. All amino acid swaps between
Mash1/MyoD or Mash1/Math1 were made by recombinant PCR
(Erlich, 1989
;
Higuchi et al., 1988
). Mash1
DNA binding mutant, Mash1NR-AQ, changed two amino acids (NR to AQ) in the
basic region similar to that previously described for Ngn1AQ
(Gowan et al., 2001
;
Sun et al., 2001
). Two
additional Mash1 DNA binding mutants (Mash1R-G and Mash1E-G) were generated
that mutated residues predicted to contact DNA based on modeling Mash1 to the
MyoD crystal structure (Ma et al.,
1994
). PCR products were digested with NcoI and
XbaI, purified, and ligated to NcoI- and
XbaI-digested pMiwIII-(myc)5 expression vector. The bHLH
domain of Math1 or Mash1 was fused to the myc-tagged VP16 activation cassette
(Triezenberg et al., 1988
) or
the myc-tagged EnR repressor domain (Smith
and Jaynes, 1996
). Note, Mash1 bHLH fusions only functioned when
Mash1 sequences were N-terminal to the VP16 or EnR cassettes. In addition,
five amino acid residues N-terminal to the Mash1 bHLH domain shown in
Fig. 4 (PAAVA) were required
for Mash1bHLH-VP16 or -EnR fusions to function. All constructs generated by
PCR were sequence verified before use, and protein expression was verified by
immunofluorescence with antibodies to the epitope tag myc or to Mash1. The
precise sequences of the bHLH regions used for the chimeric proteins and
mutants are shown.
|
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Results |
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|
Mash1 and Math1 induce formation of different neuronal populations
In a previous study, we demonstrated that Math1 and Ngn1 have distinct
roles in the specification of interneurons in the dorsal neural tube
(Gowan et al., 2001;
Helms and Johnson, 2003
). This
suggests that neural bHLH factors share a role in inducing neuronal
differentiation but have distinct roles in neuronal subtype specification. To
determine whether Mash1, like Math1 and Ngn1, has a specific role in inducing
a distinct interneuron population, we used double immunofluorescence with
antibodies to LIM homeodomain (HD) factors that have been used to identify
interneuron subtypes based on position in the dorsoventral axis, their initial
migratory patterns, and their expression of specific LIM HD transcription
factors (Fig. 2M)
(Gross et al., 2002
;
Helms and Johnson, 2003
;
Müller et al., 2002
). We
found that overexpression of Mash1 increased dI3 neurons and decreased dI1,
dI2 and dI4/6 neurons. This was detected as a 3-fold increase in Islet1 (dI3
marker) on the electroporated side (Fig.
2A,K, green), and a decrease in the number of Lhx2/9-expressing
cells (dI1) (Fig. 2A,I, red),
Lhx1/5+;Pax2- cells (dI2) (Fig.
2B,J, red) and Lhx1/5+;Pax2+ cells (dI4/6)
(Fig. 2B,L, yellow). The
increase in Islet1 was due to an increase in the dI3 population since MNR, a
marker co-expressed with Islet1 in motor neuron populations was not increased
(data not shown). Mash1-expressing progenitor cells are adjacent to dI3, dI4
and dI5 interneurons in the dorsal spinal cord
(Gross et al., 2002
;
Müller et al., 2002
).
Markers for dI5 neurons were not included in this study, and the dI4 and dI6
populations are not easily distinguishable with the markers used. As a
control, the inactive Mash1 mutant (Mash1NR-AQ) had no effect on the
composition of these interneuron populations
(Fig. 2C,D,I-L). The DNA
binding mutant Mash1R-G that appeared to inhibit neuronal differentiation (see
above), decreased dI1 and dI3 but not the Lhx1/5 containing interneuron
populations relative to the control side
(Fig. 2E,F,I-L). It is not
clear why the Lhx1/5 (dI2) population was unaffected. One possibility is that
Ngn1, which is known to be required for the dI2 population
(Gowan et al., 2001
),
efficiently binds E-box DNA as a homodimer (R.M.H. and J.E.J., unpublished
data). This is in contrast to Mash1 and Math1 that bind E-box DNA efficiently
only as heterodimers with E-proteins. Thus, the dominant negative activity of
the Mash1R-G mutant, which is probably the result of its ability to sequester
the Class II bHLH heterodimer partners (E-proteins), would not affect the
activity of the Ngn1 homodimer, and thus, dI2 interneurons would be
unaffected.
|
Math1 and Mash1 act as transcriptional activators in inducing neuronal differentiation and in specifying dorsal interneurons
Class II bHLH factors are classically reported to be transcriptional
activators. To determine whether Math1 and Mash1 were acting as activators in
the neuronal differentiation and cell-type specification functions, we
generated chimeric proteins with the bHLH domains of Math1 or Mash1 fused to
either the VP16 activation domain
(Triezenberg et al., 1988) or
the engrailed repressor (EnR) domain
(Smith and Jaynes, 1996
).
Myc-tagged control vectors containing VP16
(Fig. 3A,B) or EnR
(Fig. 3G,H), had no effect on
neuronal differentiation or alterations in cell-type composition when
electroporated into HH14-16 stage chick neural tubes. As a readout of neuronal
differentiation, we measured the fluorescence intensity of electroporated
cells in the lateral (differentiation zone) compared with that in the medial
(proliferation zone) regions (Fig.
3M,N). However, when these activator or repressor domains were
fused to the bHLH domains of Math1 or Mash1, specific phenotypes were
observed. Math1bHLH-VP16 phenocopied the activity of wild-type Math1 in both
inducing the lateral movement of cells and inducing specific cell-types to
form (Fig. 3C,D,M,N). The
electroporated cells were preferentially found lateral to the ventricular zone
suggesting a bias towards neuronal differentiation
(Fig. 3C,M), and there was a
dramatic increase in dI1 (Lhx2/9) neurons at the expense of dI3 (Islet1)
neurons (Fig. 3D,N). In
addition, Math1bHLH-EnR inhibited cells from differentiating as seen by the
electroporated cells being biased to the ventricular zone rather than being
distributed uniformly as in controls (Fig.
3 compare I with G,M). Math1bHLH-EnR preferentially decreased dI1
(Fig. 3J,N). Mash1 also acts as
an activator to induce neuronal differentiation and specify dI3 interneurons
since Mash1bHLH-VP16 phenocopied full-length Mash1, albeit not as efficiently
(Fig. 3E,F,M,N), and
Mash1bHLH-EnR inhibited neuronal differentiation, just as seen with
Math1bHLH-EnR (Fig. 3E,M). In
addition to confirming a role for Math1 and Mash1 as transcriptional
activators, these data demonstrate that sequences outside the Math1 and Mash1
bHLH domains are not required, but may modulate, their specificity of function
in this assay.
|
The basic region binds DNA and is required for the activity of bHLH transcription factors as shown here for Mash1. However, it is the Mash1 HLH domain (Fig. 4D) not the basic region (Fig. 4C) that is specifically required for Mash1 to retain its activity in this assay. Furthermore, the Mash1 HLH, not the basic region, confers onto MyoD the ability to induce cells to translocate laterally (Fig. 4I,J). Math1 HLH also confers this activity to MyoD (Fig. 4A,O). These results indicate that the HLH domains of Mash1 and Math1 contain specific information necessary to induce the lateral movement of cells in the developing chick neural tube, suggesting their importance in neuronal differentiation.
To further define which subdomain within the Mash1 HLH region is required for this activity, individual helix 1, loop and helix 2 domain swaps between Mash1 and MyoD were tested (Fig. 4E-G,K-M). The data clearly demonstrate that helix 1 of Mash1 is required for the lateral movement of the cells from the ventricular zone (Fig. 4E-G,N), and is sufficient to confer this activity to MyoD (Fig. 4K-M,O). To verify that helix 1 of Mash1 was sufficient in the context of the MyoD protein to confer multiple aspects of the neuronal differentiation phenotype, we also determined that the electroporated cells were more likely to express the neuronal marker Tuj1 than controls (Fig. 1M,Q) and had reduced potential for incorporating BrdU (Fig. 1N,R). Although this chimeric protein was not as efficient as wild-type Mash1, it still conferred this activity to MyoD. Even the Mash1 HLH domain in MyoD did not completely recapitulate the level of activity of Mash1 seen in this assay (Fig. 1Q). Furthermore, Mash1 with its helix 1 replaced by MyoD helix 1 completely lost its ability to induce neuronal differentiation (Fig. 1P,Q). Together, these data demonstrate the important domain for inducing neuronal differentiation is largely encoded in the fifteen amino acids of helix 1 of Mash1.
We also tested a subset of helix 1 amino acids for their role in this phenotype. We chose amino acids based on sequence comparison between neural bHLH factors and the non-neural MyoD bHLH. Five amino acids were tested (Fig. 4A), however, no specific amino acid was identified that was essential for the lateral movement activity of Mash1.
Helix 2 of Math1 is the critical domain for specifically inducing dI1 neurons
Helix 1 of Mash1 was identified as a necessary and sufficient domain for
induction of neuronal differentiation, an activity shared between the neural
bHLH factors. We next set out to determine if this domain, or a distinct
domain, encodes the information for the neuronal subtype specificity function
attributed to each neural bHLH factor using the dI1 and dI3 populations as the
readout. Chimeras between Math1 and Mash1 were analyzed for their effect on
the number of dI1 (Lhx2/9) and dI3 (Islet1) interneurons. Full-length Math1 or
Mash1 were modified such that subdomains of one protein were swapped with the
comparable subdomains from the other (Fig.
5A). The changes made are color coded in
Fig. 5, with Math1 amino acid
residues in green and Mash1 amino acid residues in blue. Each construct was
Myc-tagged, cloned into the pMiwIII expression vector, and electroporated into
chick neural tubes. The data are summarized in
Fig. 5A and quantification of
data is shown in the graph (Fig.
5B).
|
Further dissection of the HLH revealed that helix 2 of Math1 is both necessary and sufficient (in the context of Mash1) to increase the number of dI1 neurons. Replacing Math1 helix 2 with Mash1 helix 2 resulted in a decrease rather than increase in dI1 neurons (Fig. 5). Furthermore, Math1 helix 2 in Mash1 resulted in an increase in dI1 neurons as predicted for a Math1 phenotype (Fig. 5). This increase in dI1 neurons was not as robust as that seen with full-length Math1 but it dramatically converted the Mash1 phenotype of repressing dI1 to increasing dI1. Exchanging helix 1 or the loop region of Math1 had no effect on the cell-type specification properties of the protein (Fig. 5). Thus, Math1 helix 2 appears to be a critical domain for Math1 effects on the dI1 interneuron population.
Math1 helix 2 in the context of Mash1 was not sufficient, however, to induce a decrease in the dI3 population as was the case when the whole Math1 HLH domain was tested in Mash1. Rather, Math1 helix 2 in Mash1 caused a slight increase in the number of dI3 neurons on the electroporated side (Fig. 5). Since dI1 and dI3 both increased, we examined whether the Islet1 and Lhx2/9 populations remained in distinct populations as in controls. Using double immunofluorescence we detected interneurons inappropriately co-expressing these LIM HD factors, a situation that is not found normally (Fig. 5C). Thus, the increase in dI1 and decrease in dI3 phenotypes seen with Math1 overexpression are not always coupled, and in this case, have been separated as cells inappropriately express both markers. Taken together, although helix 2 of Math1 is the critical domain for the activity of increasing the dI1 interneuron population, the whole HLH domain of Math1 is necessary to completely switch Mash1 activity to Math1. Importantly, the HLH domain of Math1 is not sufficient in the context of the non-neural Class II bHLH MyoD to confer the complete neuronal specification program since with this chimeric protein the number of dI1 interneurons decrease instead of increase (Fig. 5).
Both helix 1 and helix 2 of Mash1 are required for controlling the number of dI3 neurons
The HLH but not the basic region of Mash1 is necessary for the increase in
dI3 neurons, and the decrease in the dI1 neurons
(Fig. 5), and furthermore, the
HLH of Mash1 is sufficient in the context of Math1 and in the context of MyoD
to alter the phenotype to the Mash1 phenotype
(Fig. 5). Further dissection of
functional domains revealed that the dI3 phenotype depends on both helix 1 and
helix 2 of Mash1. Mash1 helix 1 is necessary for the increase in the dI3
neurons but not the decrease in dI1 neurons, and it is not sufficient in the
context of Math1 to cause the increase in dI3. Likewise, Mash1 helix 2 is
necessary for the normal increase in the dI3 population seen with wild-type
Mash1, but it is not sufficient in the context of Math1 for this phenotype.
Changes in the loop region are inconsequential to the specification function
of these factors. Together these data suggest that information encoded in both
helix 1 and helix 2 is required for the specification activities of Mash1.
Modeling of Class II bHLH structures place subclass specific amino acids residues on an external surface of the dimer
In order to gain insight into how the bHLH region may function in neural
differentiation and specification, we aligned the bHLH region of known neural
bHLH proteins and MyoD. This alignment included all known species homologs and
is shown in Fig. 6A using the
mouse sequence to represent each sub-family. The alignment highlights amino
acids that are conserved globally in all Class II bHLH factors (red), and
those that are conserved only within a specific sub-family (blue). The
conserved amino acids were then mapped onto an to a computer generated 3D
structure of Mash1, modeled on the 3D crystal structure of MyoD
(Ma et al., 1994) (see
Materials and methods). This reveals a clear difference in the spatial
distribution of the two types of conserved amino acids. The globally conserved
residues are positioned at either the DNA or E-protein dimerization interface
(Fig. 6B). In contrast, the
conserved residues specific to a sub-family, project away from these
interfaces creating surfaces that are accessible to potential co-factors.
|
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Discussion |
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Shared versus distinct functions of the neural bHLH factors in inducing neuronal differentiation and cell-type specification in the spinal neural tube
Mash1, Math1 and Ngn1 are expressed with similar timing in ventricular zone
cells in the developing neural tube. However, their expression spatially is in
non-overlapping patterns in these progenitor cells along the dorsoventral axis
(Gowan et al., 2001;
Ma et al., 1997
). Consistent
with these expression characteristics, we have identified both shared and
distinct functions for Mash1, Math1 and Ngn1
(Gowan et al., 2001
). Their
common function is in their ability to induce neuronal differentiation when
each neural bHLH factor is overexpressed in ventricular zone cells within the
chick neural tube. This over-expression biases the cells to exit the cell
cycle, move laterally out of the ventricular zone, and initiate expression of
neuronal-specific genes within 24 hours. This function in inducing
neurogenesis has been previously ascribed to these neural bHLH factors using
multiple paradigms including mis-expression in retina and cortex
(Cai et al., 2000
;
Cepko, 1999
), in cultured
cortical progenitors (Sun et al.,
2001
), and in P19 embryonal carcinoma cells
(Farah et al., 2000
). Together
with in vitro binding data that show the neural bHLH factors can bind similar
DNA sequences (Bertrand et al.,
2002
), it is probable that there are shared downstream targets to
carry out this function in neuronal differentiation. Indeed, shared downstream
targets, whether direct or indirect, have recently been shown for the
Xenopus homologs Xash1 and Xngnr1
(Talikka et al., 2002
).
In addition to the shared function in inducing neuronal differentiation,
distinct functions for Mash1, Math1 and Ngn1 have been identified. Each factor
specifies a different neuronal sub-type in chick neural tube in this assay.
This activity is best distinguished by following the three most dorsal
interneuron populations dI1, dI2 and dI3
(Helms and Johnson, 2003).
Math1 induces and is required for the formation of dI1 neurons
(Bermingham et al., 2001
;
Gowan et al., 2001
). Ngn1
induces, and with Ngn2, is required for the formation of dI2 neurons
(Gowan et al., 2001
). And
finally, although some dI3 neurons can form in the absence of Mash1 (A.W.
Helms and J.E.J., unpublished data), Mash1 can induce dI3 neurons. In each
case, the increase in one population appears to occur at the expense of the
other two. This may reflect the fact that each bHLH represses the expression
of the other bHLH factors in the dorsal neural tube
(Gowan et al., 2001
).
Specificity in function has been demonstrated for neural bHLH factors in
multiple regions of the developing nervous system including the forebrain
(Fode et al., 2000
;
Parras et al., 2002
), the
neural crest (Perez et al.,
1999
) and retina (Perron et
al., 1999
). In addition, downstream targets have been identified
for the Xngnr1 pathway that are not shared with Xash1
(Talikka et al., 2002
). The
distinct functions demonstrated for Mash1, Math1 and Ngn1 suggest that each
factor has distinct downstream targets, and since these factors have different
activities in different regions of the nervous system, these targets are
likely context dependent.
Mash1 progenitor cells are located adjacent to the dI3, dI4 and dI5 interneuron populations, and it has been supposed that there is a lineage relationship between these cells. In this context, it was surprising that over-expression of Mash1 resulted in a moderate decrease in dI4/6 interneurons as defined by Lhx1/5 and Pax2 expression. Since dI4 and dI6 are difficult to distinguish by markers at this time, it is not clear what the effect is specifically on dI4. However, the absence of an increase in dI4 may suggest the progenitor/interneuron relationship between Mash1-expressing progenitor cells and dI4 interneurons should be revisited.
Protein-protein interaction domains of Mash1 and Math1 are required for their specific activities
Mash1 and Math1 are class II bHLH factors that characteristically have
tissue-specific expression, and bind E-box DNA (CANNTG) as heterodimers with
class I bHLH factors such as E12, E47, HEB and E2-2
(Massari and Murre, 2000).
Crystal structures of a non-neural class II bHLH factor MyoD, or a class I
bHLH factor E47, demonstrated that the basic region interacts with DNA and the
HLH forms an amphipathic helix that is involved in protein-protein
interactions in the formation of homo- or heterodimers
(Ellenberger et al., 1994
;
Ma et al., 1994
). Studies with
other bHLH factors have demonstrated the importance of the basic region for
specific functions (Chien et al.,
1996
; Davis and Weintraub,
1992
; Dezan et al.,
1999
). More recently, however, studies of the Xenopus
neural bHLH factors Xash1 (Mash1 homolog) and Xngnr1 (Ngn homolog) identified
the HLH domain as the region encoding information for induction of specific
downstream targets (Talikka et al.,
2002
). Our findings are similar to these latter experiments,
demonstrating that the HLH domain, and not the basic region, encodes the
necessary information for the specific functions in neurogenesis of the neural
bHLH factors Mash1 and Math1 in the chick neural tube.
Since the HLH domain functions in protein-protein interactions, it is
reasonable to propose that specificity of function may be conferred by
interactions with specific cofactors that vary between the different bHLH
proteins. It is known that in vitro, DNA binding activity is most efficient
with heterodimers of the neural bHLH with an E-protein such as E12
(Akazawa et al., 1995;
Helms et al., 2000
;
Johnson et al., 1992
). There
are no reports that the individual E-proteins (E2a, HEB, E2-2) can confer
specificity of function on the neural bHLH/E-protein heterodimer, and knockout
studies with the E-protein genes suggest they have a high level of functional
redundancy (Zhuang et al.,
1998
; Zhuang et al.,
1996
). Thus, it may be that context-dependent co-factors that form
higher order complexes with bHLH heterodimers are important for specific
functions. Further support for the importance of cellular context for bHLH
factor function is provided by the chick electroporation experiments. For
example, electroporation of pMiWIII-Math1 results in overexpression of Math1
along the extent of the dorsoventral axis, however, the increase in the dI1
interneurons is biased to the dorsal regions. This is in contrast to the
neuronal differentiation phenotype that is seen throughout the dorsoventral
axis. Invoking context-dependent protein-protein interactions to explain the
specificity of bHLH function along the dorsoventral axis of the spinal neural
tube is also helpful in explaining how bHLH factors are required for different
types of neurons in different regions of the nervous system. Consistent with
the involvement of specific co-factor interactions is the presence of
conserved surface amino acids within each neural bHLH sub-family that are
distinct between the different sub-families
(Fig. 6).
Besides the E-proteins, a number of other co-factors have been described
that form complexes with Class II bHLH factors. In Drosophila, Chip,
a LIM homeodomain binding protein (homolog of Ldb factors in vertebrates), has
been shown to form a complex with Achaete (Mash1 homolog) and Daughterless
(E12 homolog), possibly as an adapter protein bringing another transcription
factor, Pannier, in to form a higher order transcriptional complex
(Ramain et al., 2000). In
studies of Tal1, a class II bHLH factor important in hematopoiesis, a LIM only
factor (LMO) and Ldb factors were seen to form a complex with Tal1 and E12
(Wadman et al., 1994
;
Wadman et al., 1997
). In the
neural tube, Pfaff and colleagues have demonstrated that higher order complex
formation of homeodomain factors and bHLH factors control ventral cell fates.
In this case, the homeodomain factor Lhx3 alone is not sufficient to generate
motor neurons, but in combination with Ldb it specifies V2 interneurons. When
co-expressed with islet1, a higher order complex is formed, resulting in
different DNA binding characteristics, and the specification of motor neurons
rather than V2 interneurons (Thaler et
al., 2002
). Furthermore, two bHLH factors, Ngn2 and NeuroM,
transcriptionally synergize with the homeodomain complex to specify the motor
neurons (Lee and Pfaff, 2003
).
This type of combinatorial interactions of transcription factors is an
attractive hypothesis for the context-dependent functions seen with the bHLH
factors studied here for dorsal neural tube development. The identity and role
of co-factors in forming higher order transcriptional complexes with Mash1 and
Math1 is yet to be determined.
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ACKNOWLEDGMENTS |
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