1 Department of Neurobiology and Anatomy, University of Utah School of Medicine,
Salt Lake City, UT 84132, USA
2 Divisions of Developmental Biology and Ophthalmology, Children's Hospital
Research Foundation, Cincinnati, OH 45229, USA
* Author for correspondence (e-mail: monica.vetter{at}neuro.utah.edu)
Accepted 16 December 2004
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SUMMARY |
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Key words: Ath5, Math5 (Atoh7), Retina, Development, Regulation, Transgenic, Xenopus
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Introduction |
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Ath5, a vertebrate homolog of Drosophila atonal, is
expressed in the developing retina in all vertebrates studied and plays a
crucial role in regulating retinal neurogenesis
(Vetter and Brown, 2001). In
Xenopus, Xath5 is expressed in a tightly restricted set of cells in
the developing neural retina, the olfactory placodes and the pineal gland. In
the retina, Xath5 expression commences in retinal progenitors just
prior to cell cycle exit and onset of differentiation, but expression is
downregulated before cells become fully mature retinal neurons
(Kanekar et al., 1997
). In
mouse, zebrafish and chick, Ath5 is also expressed in a similar
restricted manner within the retina immediately preceding the onset of retinal
ganglion cell (RGC) differentiation (Brown
et al., 1998
; Kay et al.,
2001
; Matter-Sadzinski et al.,
2001
). Ath5 is specifically required for the
differentiation of the RGC cell type as in both mouse and zebrafish
Ath5 loss-of-function mutants, RGCs are either drastically reduced or
missing altogether (Brown et al.,
2001
; Kay et al.,
2001
; Wang et al.,
2001
).
As Ath5 plays such a key role in retinal development, it is
important to understand how its expression is regulated. Analysis of the
chicken Ath5 (Cath5) promoter has suggested that it can be
regulated by multiple bHLH factors in retinal cell culture, and that both
Cath5 and Ngn2 are bound to the Cath5 promoter at specific stages of
retinal development (Matter-Sadzinski et
al., 2001; Skowronska-Krawczyk
et al., 2004
). However, it is not yet known what is required for
correct tissue-specific expression in vivo. Recent genome sequencing efforts
have led to the development of phylogenetic footprinting strategies, whereby
cross-species sequence comparison of noncoding regions from homologous genes
can identify candidate enhancers that may play a role in regulating gene
expression. This approach provides excellent predictive power for functional
enhancer elements (Bulyk,
2003
); however, all candidate elements must ultimately be tested
in vivo to determine whether they can regulate gene expression and assess how
they contribute to the normal pattern of expression during development
(Muller et al., 2002
).
In this study, we have analyzed the regulation of Ath5 expression in Xenopus, as well as in mouse. First, we identified a highly conserved proximal non-coding region, and showed that it mediates bHLH-dependent regulation of Ath5 expression. This region was sufficient to promote transgene expression in Xenopus, but not in mouse. Second, we show that a longer transgene, that includes additional 5' cis-regulatory sequence, promotes bHLH-independent transgene expression in Xenopus. Longer transgenes from either the mouse or Xenopus Ath5 cis-regulatory region were also sufficient to promote expression in mouse. For the Math5 transgene (Atoh7 Mouse Genome Informatics), expression did not depend upon Math5 itself. Thus, there exist both bHLH-dependent and-independent modes of Ath5 gene regulation; however, the importance of bHLH-dependent expression mediated through the conserved proximal region may be species dependent.
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Materials and methods |
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Transgenic procedure
The generation of transgenics was carried out as described
(Kroll and Amaya, 1996), with
variations (Hutcheson and Vetter,
2002
). Whole-mount embryos were analyzed for GFP expression
between stages 28 and 42, when the majority of retinal neurons are born.
Embryos were scored as retinal positive if there was specific retinal
expression that was similar in pattern and timing to endogenous Xath5
expression. For constructs with weak or no detectable GFP expression by
fluorescence, expression or lack of expression was confirmed either by in situ
hybridization or by whole-mount antibody staining using a polyclonal anti-GFP
antibody (Torrey Pines) and an Alexa-Fluor 488-conjugated secondary antibody
(Molecular Probes). For analysis on sections, embryos were sectioned on a
cryostat at a thickness of 14 µm.
In situ hybridization
Embryos were processed for in situ hybridization as described previously
(Kanekar et al., 1997). Double
in situ hybridization on sections was performed using a digoxigenin-labeled
GFP probe and a fluorescein-labeled Xath5 probe as previously
described (Hutcheson and Vetter,
2001
).
Sequence analysis
Sequence analysis was performed using VISTA
(http://www-gsd.lbl.gov/vista/)
(Frazer et al., 2004;
Mayor et al., 2000
). Alignment
criteria used were 80% nucleotide identity over 30 bp blocks. Candidate
transcription factor binding sites were identified using the Genomatix
MatInspector module
(http://www.genomatix.de/)
(Quandt et al., 1995
), the
Transfac database
(www.gene-regulation.com)
(Wingender et al., 1996
) or
ClusterBuster
(http://zlab.bu.edu/cluster-buster/)
(Frith et al., 2003
).
Other vertebrate Ath5 genomic sequences were identified as
follows: X. tropicalis
(http://genome.jgi-psf.org/xenopus0/xenopus0.home.html);
mouse and human Ath5 (Atoh7) have been previously isolated
and aligned (Brown et al.,
2002); chick (GenBank Number AJ630209)
(Skowronska-Krawczyk et al.,
2004
); Fugu (scaffold 1775;
http://genome.jgi-sf.org/fugu3/fugu3.home.html);
and zebrafish (clone AL627094;
http://trace.ensembl.org/perl/ssahaview).
The genomic sequence for Drosophila atonal was derived from BAC clone
#AC008094
(http://flybase.bio.indiana.edu/).
Generation of transgenic mice
0.6 kb and 2.3 kb sequences found immediately upstream from the
Math5 start ATG codon (nucleotides 2472 to 3072 and 772 to 3072 from
AF418923) were cloned into the pG1 GFP reporter construct. These
Math5 (M5) enhancer-promoters and the GFP-coding sequence were
purified away from vector sequences and used to generate independent mouse
lines transgenic with either the M5-0.6 kb or M5-2.3 kb transgene. Similarly,
the Xenopus pG1X5-proximal or pG1X5-3.3 kb constructs were used to
generate transgenic mice. All transgenic strains are viable and fertile and
maintained on a CD1 background. For some experiments the M5-2.3 transgenic
line was crossed with the Math5 mutant
(Brown et al., 2001) maintained
on CD-1. E12.5 or E13.5 mouse embryos from timed pregnancies were harvested,
kept on ice in PBS for whole mount imaging. The genotype of
M5-2.3Tg/+ or M5-2.3Tg/+;Math5/
embryos was determined by PCR genotyping of embryonic or adult tail DNA using
primers specific for GFP coding sequences or as described previously
(Brown et al., 2001
).
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Results |
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Isolation of a 201 bp enhancer region
To define which regions within the 3.3 kb Xath5 cis-regulatory
region are crucial for retinal expression, we generated a series of deletion
constructs (pG1X5-HindIII, pG1X5-
DraII, and
pG1X5-
PstI) by restriction digest
(Fig. 2A). When each construct
was tested in transgenic Xenopus embryos, expression of the GFP
transgene was comparable with that seen with the full pG1X5-3.3 kb construct
(Fig. 2A). Thus, the 464 bp of
sequence closest to the Xath5-coding region are sufficient to promote
transgene expression in the retina. To verify this, the pG1X5-proximal
construct containing only 427 bp of upstream sequence was used to generate
transgenic embryos (Fig. 2B).
These embryos consistently expressed GFP in the retina
(Fig. 2C). Expression in the
olfactory placodes and pineal was also observed, but less frequently (data not
shown).
|
To assess whether the transgene is expressed in the appropriate cells
within the retina, we performed double in situ hybridization on sections from
stage 42 pG1X5-proximal transgenic embryos, and found that the expression of
GFP RNA (Fig. 3A,E)
was restricted to the ciliary marginal zone (CMZ) in a pattern identical to
endogenous Xath5 mRNA (Fig.
3C,G) (Kanekar et al.,
1997). Interestingly, at stage 42, GFP fluorescence was visible
throughout the central retina in all three neuronal layers and in most
neuronal cell types, probably owing to perdurance of the GFP protein
(Fig. 3D). This is consistent
with recent lineage analysis of Math5-expressing cells in the mouse
retina showing that they give rise to all major classes of retinal cell types
that occupy the three cell layers (Yang et
al., 2003
) (J. Brzezinski and T. Glaser, unpublished).
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To further test whether expression of the pG1X5-proximal transgene depends upon bHLH activity, we generated transgenic embryos then injected RNA for RFP (red fluorescent protein; 1 ng) and a dominant-negative form of Xath5 (500pg) into a dorsal animal blastomere at the eight-cell stage. Dominant-negative Xath5 was created by replacing the putative Xath5 activation domain with the repressor domain from Drosophila Engrailed to create Xath5-EnR. This dominant-negative protein interferes with the proneural activity of multiple atonal-related bHLH factors, including Xath5, Xath3, NeuroD and XNgnR1 (T. VanRaay, M. Logan and M.L.V., unpublished). Expression of Xath5-EnR was able to suppress pG1X5-proximal transgene expression on the injected side (6/13; Fig. 5C-E), arguing that proneural bHLH activity is required for expression driven by the proximal Xath5 cis-regulatory region.
E-boxes, together with adjacent conserved sequences, are sufficient to promote transgene expression in the retina
Although E1 and E2 are necessary elements in the proximal promoter, we
sought to define the minimal set of elements sufficient to drive retinal
specific expression of the transgene. To test the E-boxes alone, two copies of
E1 and E2 (272 to 253) were fused to the pG1X5-TATAA construct
(Fig. 5F). The resulting
transgene pG1X5-TATAA+2xE1E2 never promoted detectable GFP expression. Thus,
the highly conserved E-boxes alone are insufficient for transgene expression.
We then created pG1X5-TATAA+33 bp (Fig.
5F), which includes one copy of E1 and E2 as well as an adjacent
motif that resembles the -box previously identified in the
Drosophila scute sensory mother cell (SMC) enhancer
(Culi and Modolell, 1998
). In
Xenopus embryos transgenic for pG1X5-TATAA+33 bp, GFP expression was
not specific and was found in a wide range of neural and muscle tissues
(Fig. 5G).
Next we created pG1X5-TATAA+48 bp, which includes an additional 15 bp of conserved upstream sequence (Fig. 5F). Embryos transgenic for pG1X5-TATAA+48 bp showed GFP expression that was strikingly similar to endogenous Xath5 expression, and also included consistent weak expression in axial muscles (Fig. 5F,H), with a small percentage of embryos showing only axial muscle expression. We conclude that addition of this 15 bp region is sufficient to confer almost complete tissue specificity to transgene expression. Interestingly, this region is highly conserved across species (11/15 nucleotides identical between Xenopus laevis and mouse), but contains no known transcription factor binding sites.
This 48 bp fragment contains two E boxes, the -box and the
additional 15 bp fragment required for tissue-specific expression. To assess
whether the
-box contributes to the strength or specificity of
expression, we mutated four residues within the
-box sequence
(pG1X5-TATAA+48 bp-
mut). We found that this construct yielded a
significantly reduced fraction of GFP positive transgenic embryos, but the
overall pattern was unchanged (Fig.
5F). Thus, the
-box appears to contribute to the strength
but not specificity of expression promoted by the short 48 bp fragment.
bHLH-independent regulation of 3.3 kb Xath5 transgene expression
To test whether bHLH factors in Xenopus can regulate
Xath5 transgene expression in vivo, we overexpressed proneural bHLH
factors in pG1X5-3.3 kb transgenic embryos by fertilizing eggs from an adult
transgenic female and injecting RNA at the eight-cell stage encoding for RFP
(1 ng) and for either Xath5, XngnR1, Xath3, XNeuroD, Xash1 or Xash3 (500 pg
each). No change in transgene expression was observed with overexpression of
RFP alone (21/21; Fig. 6A-C).
We observed significant ectopic GFP expression with overexpression of the
atonal-related bHLH factors Xath5 (30/30), XngnR1 (26/28), Xath3 (9/11) or
NeuroD (12/12; Xath5 shown in Fig.
6D-F, and data not shown), but not with overexpression of either
Xash1 (50/50) or Xash3 (14/15), which are Achaete-Scute-related factors (data
not shown). Thus, the pG1X5-3.3 kb transgene, which includes the conserved
proximal region, can be recognized and activated by Atonal-related bHLH
factors.
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As there are additional non-conserved E-boxes, we wanted to further test whether bHLH activity is required for pG1X5-3.3 kb transgene expression using Xath5-EnR. We fertilized eggs from an adult Xenopus laevis female transgenic for the pG1X5-3.3 kb transgene, and injected RNA at the eight-cell stage encoding for Xath5-EnR (500 pg) and for RFP (1 ng). Overexpression of Xath5-EnR did not suppress pG1X5-3.3 kb transgene expression (63/63; Fig. 6H-J), and also did not suppress expression of endogenous Xath5 (15/16; data not shown). Together, these data argue for a bHLH-independent component to Xath5 gene regulation in vivo, probably mediated by more distal cis-regulatory sequences.
A distal regulatory region in the Ath5 gene is sufficient for retinal expression
To test whether distal cis-regulatory sequences alone have enhancer
activity, we fused a distal PstI fragment that lacks the conserved
proximal region (3162 to 464) to pG1X5-TATAA. We observed
specific retinal GFP expression in 39% of transgenic embryos
(Fig. 7A,B), with much less
frequent olfactory and/or pineal expression as well (data not shown). This
distal region also promoted weak but specific retinal expression when coupled
to the mouse Fos basal promoter (Fig.
7A). Thus, the distal Xath5 cis-regulatory region
contains elements sufficient for retinal expression that are distinct from the
conserved proximal region.
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In the developing chick retina, Cath5 is bound to its own promoter
(Skowronska-Krawczyk et al.,
2004), suggesting that autoregulation may play a role in
Ath5 gene regulation. In addition both Atonal and Math1 (Atoh1)
regulate their own expression (Baker et
al., 1996
; Helms et al.,
2000
). Therefore, we tested for autoregulation of Math5
by crossing two independent 2.3 kb M5-GFP transgenic lines to mice carrying
the Math5 targeted deletion (Brown
et al., 2001
). Mice heterozygous for both the transgene and mutant
allele were intercrossed and the resulting embryos examined for GFP
fluorescence in the optic cup at E13.5, when endogenous Math5
(Brown et al., 1998
) and the
2.3 kb M5-GFP transgene are maximally expressed. This experiment
was performed four independent times, using two litters for each transgene
(n=56 embryos total). In every mutant embryo that possessed the
transgene (n=12), robust GFP fluorescence was observed (compare
Fig. 8F with 8G). Thus, Math5
is not required for transgene expression, suggesting that autoregulation is
not an essential mechanism for regulating Math5 expression during
mouse retinal development.
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Discussion |
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Activity of the Xath5 proximal cis-regulatory region is bHLH-dependent
bHLH factors probably play a role in regulating the retinal expression of
Xath5 in vivo through the conserved proximal region. There is ample
precedence for vertebrate bHLH factor cross-regulation and autoregulation. For
example, in Xenopus during primary neurogenesis at open neural plate
stages, Xngnr-1 activates the expression of Xath3 and
NeuroD, while Xath3 and NeuroD crossactivate each other's expression
(Ma et al., 1996;
Perron et al., 1999
).
Expression of a 1 kb Neurod2 promoter in mouse brain was also
recently shown to depend upon two E boxes in the proximal promoter region
(Lin et al., 2004
).
We found that overexpression of multiple atonal-related bHLH factors,
including Xath5, Xath3, XNeuroD and X-ngnr-1, could ectopically activate
pG1X5-3.3 kb transgene expression in Xenopus embryos. Which of these
are likely to play a role in regulating Xath5 expression in vivo? In
the CMZ, a number of bHLH factors are expressed in an overlapping but
sequential manner in a peripheral to central spatial pattern that reflects the
sequence of gene activation during early eye development
(Perron et al., 1998).
XNeuroD and Xath3 (NeuroM) do not precede
Xath5 (Kanekar et al.,
1997
; Perron et al.,
1999
), so they are unlikely to initiate Xath5 expression,
but could help maintain retinal expression. X-ngnr-1 is expressed in
the CMZ in an earlier yet overlapping pattern, so it may help initiate and/or
maintain Xath5 expression. In chick, the related gene Ngn2
is co-expressed with Cath5 in early retinal progenitors, and Ngn2
protein is bound to the Cath5 promoter during retinal development,
and activates expression of a Cath5 transgene in cell culture
(Skowronska-Krawczyk et al.,
2004
; Matter-Sadzinski et al.,
2001
). Thus, X-ngnr-1 (or Ngn2 in chick) is a strong candidate for
regulating Ath5 expression in vivo.
We did not observe ectopic pG1X5-3.3 kb transgene expression with
overexpression of either Xash1 or Xash3, suggesting that these factors do not
positively regulate Xath5 gene expression. In chick, Ath5
and Ash1 are expressed in mutually exclusive domains in the retinal
neuroepithelium, and Cash1 expression inhibited activity of the chick
Ath5 regulatory region in transfected retinal cells in culture
(Matter-Sadzinski et al.,
2001). It remains possible that in Xenopus, Xash1 or
Xash3 could have similar inhibitory activity on bHLH-dependent expression from
the pG1-X5 proximal region alone.
E boxes alone are not sufficient for retina-specific expression
We determined that either E1 or E2 are necessary in the context of the
proximal Xath5 promoter to drive retinal expression of a GFP
transgene. However these two highly conserved and essential E-boxes alone were
insufficient for retinal-specific expression. A 33 bp fragment that included
E1 and E2 plus adjacent conserved 5' sequence drove GFP expression
throughout the CNS as well as in muscle, suggesting it may be non-specifically
recognized by multiple bHLH factors. This 33 bp sequence includes a motif that
is similar to the consensus for an -box, which was first identified as
a critical element in the scute sensory mother cell (SMC) enhancer
(Culi and Modolell, 1998
), and
may be a candidate binding sequence for a winged-helix zinc finger
transcription factor (Genomatix MatInspector). In Xenopus, this
sequence played a role in robustness but not specificity of transgene
expression.
Inclusion of an additional 15 bp of 5' sequence constrained transgene expression to tissues where endogenous Xath5 is expressed, along with weak expression in the axial muscles. The additional 15 bp of sequence included in this construct are highly conserved, with 11 out of 15 nucleotides identical between Xenopus laevis and mouse; however, no candidate transcription factor binding sites were apparent. It is intriguing to speculate that some novel factor interacts with this sequence and participates in bHLH-dependent Ath5 gene regulation.
Proximal Ath5 sequences are not sufficient for gene expression in mouse
In the mouse retina, bHLH factors are unlikely to initiate Math5
expression. Ngn2 commences expression at E13, 2 days later than
Math5 (Brown et al.,
1998), and Math5 is not preceded by expression of genes
encoding any other known proneural bHLH factor. Thus, Math5 must be
regulated by other factors present in the developing optic vesicle and cup.
Consistent with this idea, the short proximal region from either mouse or
Xenopus, which contains the conserved E-boxes, was insufficient to
promote transgene expression in mouse. Thus, despite strong sequence homology
in the proximal cis-regulatory region, the role bHLH factors play in
regulating the expression of Ath5 appears to be species specific.
This may in part depend upon whether other bHLH factors precede Ath5,
as in frog and chick, or whether it is the first retinal bHLH gene expressed,
as in the mouse. We also cannot rule out a role for bHLH factors in some other
aspect of Ath5 gene regulation, potentially at later stages of
development in mouse. It also remains possible that in Xenopus or
chick, bHLH factors do not play an essential role in regulating Ath5
gene expression as we identified a bHLH-independent distal cis-regulatory
region that alone was sufficient to promote retinal transgene expression.
Is there autoregulation of Ath5 gene expression?
Autoregulation may also play a role in regulating Xath5
expression. In Drosophila, Atonal function is crucial for its own
expression (Sun et al., 1998),
and in vertebrates Math1 has autoregulatory activity in the developing spinal
cord, but not in the cerebellum (Gazit et
al., 2004
; Helms et al.,
2000
). In chick retinal cultures, Cath5 transgene
expression could be activated by chick Ath5 itself and Cath5 was found bound
to its promoter in vivo, suggesting autoregulation
(Skowronska-Krawczyk et al.,
2004
; Matter-Sadzinski et al.,
2001
). Consistent with this, we showed that overexpression of
Xath5 could activate ectopic transgene expression in Xenopus.
However, in mouse expression of the pG1M5-2.3 kb transgene did not depend upon
Math5, arguing that autoregulation is not essential for Ath5 gene
expression in the developing mouse retina. This fits with the observation that
expression of a ß-galactosidase reporter introduced into the
Math5 locus by homologous recombination is expressed in
Math5/ optic cup and retina (E11-birth)
(Brown et al., 2001
) (data not
shown). Consistent with this, expression of the longer pG1X5-3.3 kb transgene
in Xenopus, as well as expression of endogenous Xath5, was
not blocked by the dominant-negative Xath5-EnR. Thus, there are clearly
additional sequences that can mediate Ath5 gene expression in both
species, as discussed below. This outcome does not preclude the possibility
that autoregulation contributes to some aspect of Ath5 expression
during retinal development.
bHLH-independent gene regulation mediated through distal cis-regulatory sequences
We found that that unlike the proximal Xath5 transgene, expression
driven by the full 3.3 kb transgene was not bHLH dependent, as it did not
require four conserved E-boxes and could not be suppressed by overexpression
of Xath5-EnR. Thus, we conclude that more distal sequences mediate
bHLH-independent expression. We found that a distal fragment from the
Xath5 cis-regulatory region alone was sufficient to promote retinal
transgene expression in Xenopus. Future analysis will focus on
defining which elements within this region are required for expression.
Interestingly, longer 5' cis-regulatory regions from either mouse (2.3
kb) or Xenopus (3.3 kb) were sufficient to promote appropriate
transgene expression in mouse, consistent with our conclusion that
Ath5 gene expression in mouse is largely bHLH-independent. However,
there is little sequence homology between mouse and Xenopus in the
distal region other than E3 and E4, so it is unclear whether the mechanisms
governing Math5 and Xath5 gene regulation through more
distal sequences will be conserved.
What are the signals that regulate bHLH-independent Ath5 gene
regulation? In Drosophila, the initiation and propagation of
atonal expression directly depends upon signals such as hedgehog
(Dominguez, 1999;
Sun et al., 1998
). However in
the 3.3 kb Xath5 cis-regulatory sequence, we found no clear binding
sites for Gli zinc finger transcription factors, which mediate hedgehog
signaling, although it remains possible that regulation is indirect. In the
fish retina, Ath5 expression is initiated by a signal from the optic
stalk (Masai et al., 2000
),
and it is possible that this signal acts through the cis-regulatory sequences
that we have identified. In support of this idea, the 3.3 kb pG1X5 transgene
was expressed in the developing retina in transgenic zebrafish embryos,
suggesting that the 3.3 kb Xath5 regulatory sequences are
appropriately recognized in zebrafish (A. Pittman and C.-B. Chien, personal
communication).
bHLH-dependent and -independent Ath5 gene regulation
We have identified bHLH-dependent and -independent modes of Ath5
gene regulation in Xenopus, raising the issue of how they contribute
to endogenous Xath5 expression. It is possible that the distal and
proximal cis-regulatory sequences serve overlapping or redundant functions.
Alternatively, the distal and proximal regions may regulate different phases
of Xath5 expression. For example, during Drosophila eye
development atonal gene expression is initiated in a bHLH-independent
fashion by factors such as hedgehog, then expression becomes dependent upon
Atonal itself (Hsiung and Moses,
2002). It is therefore possible that Xath5 gene
regulation is similar, with initiation of gene expression being
bHLH-independent and maintenance of expression requiring Xath5 and/or other
bHLH factors such as X-Ngnr-1. In chick, bHLH factors are clearly involved in
regulation of Cath5 (Skowronska-Krawczyk
et al., 2004
; Matter-Sadzinski
et al., 2001
), but it remains to be determined whether there is
bHLH-independent regulation as well. In mouse, we found no evidence for
bHLH-dependent Atoh7 gene regulation, demonstrating that
Math5 expression is bHLH independent. Thus, we have shown that
although some mechanisms of Ath5 gene regulation are conserved, there
are intriguing species-specific differences that remain to be explored.
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ACKNOWLEDGMENTS |
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