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First published online 13 December 2006
doi: 10.1242/dev.02734
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Biology Department, Texas A&M University, College Station, TX 77843-3258, USA.
Author for correspondence (e-mail:
briley{at}mail.bio.tamu.edu)
Accepted 31 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hair cells, Proneural genes, Fgf, Delta-Notch, Pax2-5-8, Foxi1, Dlx, Msx, no isthmus (pax2a), mind bomb
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). As
the equivalence group develops, a few cells upregulate Atoh1
expression and complete differentiation as hair cells. The rest lose
expression of Atoh1 and become support cells. As the principal
regulator of hair cell differentiation, Atoh1 has received great
attention in recent years in both basic and applied research
(Shailam et al., 1999;
Lanford et al., 2000;
Zheng and Gao, 2000;
Itoh and Chitnis, 2001;
Chen et al., 2002;
Wang et al., 2002;
Woods et al., 2004;
Fritzsch et al., 2005;
Izumikawa et al., 2005;
Kelley, 2006). However,
despite extensive analysis of Atoh1, a number of fundamental issues
still need to be resolved. Most notably, there are conflicting reports as to
the precise role(s) of Atoh1 in otic development. Although
Atoh1 is maintained only in hair cells, it may function earlier to
specify the equivalence group itself - a definitive proneural function.
Accordingly, disruption of mouse Atoh1 (Math1) ablates all
hair cells and support cells in the cochlea
(Woods et al., 2004). However,
the persistence of cells expressing some early markers of sensory epithelia
has been interpreted to mean that mouse Atoh1 is not required for
specifying the equivalence group per se, but instead only promotes the final
stages of hair cell development (Bermingham
et al., 1999; Chen et al.,
2002; Fritzsch et al.,
2005). Additionally, a key aspect of prosensory development does
not require Atoh1: prospective sensory cells begin to express
p27kip1 and exit the cell cycle before expression of
Atoh1, and this process still occurs in Atoh1 mutants.
However, p27kip1 expression and cell cycle withdrawal
could be regulated independently from equivalence group specification. Indeed,
sensory epithelia still form in p27kip1 mutants, despite
the failure of cells to properly exit the cell cycle
(Chen and Segil, 1999). This
leaves open the question of when the equivalence group forms and whether
Atoh1 acts early or late in the process.
Work on Drosophila ato provides a useful paradigm for testing
vertebrate Atoh1 function (Fig.
1). ato is initially expressed in a broad pattern (the
equivalence group) well before cell fate specification
(Jarman et al., 1995). The
equivalence group then restricts its own size through activation of
Delta-Notch (Dl-N) signaling (Baker et al.,
1996; Baker and Yu,
1997). In this process, N-dependent downregulation of ato
breaks the equivalence group into discrete `intermediate groups' of
ato-expressing cells separated by non-expressing cells that are
excluded from the sensory structure. Subsequently, the balance of ato and N
activity selects between alternate fates within intermediate groups (lateral
inhibition). Because ato is required for Dl expression,
ato mutants fail to activate N-mediated restriction of ato,
resulting in retention of a broad field of ato-expressing cells that
are otherwise blocked from further development
(Jarman et al., 1995;
Baker and Yu, 1997). Similarly,
N mutants also fail to restrict ato expression, but in this
case all cells differentiate as sensory cells
(Baker et al., 1996).
Paradoxically, during the prosensory phase of development elevating N activity
by expressing N intracellular domain (NICD) enhances ato expression
(Baker and Yu, 1997). This
involves a poorly characterized branch of the N pathway not requiring Su(H)
(Ligoxygakis et al., 1998).
During subsequent phases of development, NICD activates the canonical N
pathway and abolishes ato expression. This work provides clear
predictions for how vertebrate Atoh1 might function, assuming it acts
as a classic proneural gene. By contrast, terminal differentiation factors
such as NeuroD are insensitive to N activity and are not required for cell
fate specification (Chitnis and Kintner,
1996) (reviewed by Brunet and
Ghysen, 1999; Hassan and
Bellen, 2000).
A crucial determinant of proneural gene function is the regulatory context
in which it operates (Niwa et al.,
2004). Activation of ato requires combinatorial signaling
and specific regional identity genes such as eyeless (Pax6),
which also modify the sensory fate specified by ato
(Niwa et al., 2004). The
factors that induce Atoh1 in the ear and cooperate in its function
are largely unknown. Sox2 is expressed broadly in the early otic
vesicle in mouse and is required for induction of Atoh1 several days
later (Kiernan et al., 2005b).
The lag in Atoh1 expression suggests that Sox2 works combinatorially
with other factors to initiate prosensory development. A number of signaling
molecules have also been implicated in sensory epithelium development
(Pirvola et al., 2002;
Stevens et al., 2003;
Daudet and Lewis, 2005;
Brooker et al., 2006;
Kiernan et al., 2006;
Pujades et al., 2006), but
their relationships to Atoh1 expression remain unknown. Identifying
the upstream activators of Atoh1 is essential for understanding the
regulatory network leading to formation and maintenance of hair cells.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Itoh et al.,
2003). b380 is a deletion of dlx3b and
dlx4b and mutants are easily identified after 1l hours
post-fertilization (hpf) by lack of somitic segmentation
(Fritz et al., 1996). The
hsp70-dnSu(H) line was developed by Latimer et al.
(Latimer et al., 2005), and
the hsp70-Gal4 and UAS-NICD lines were developed by Scheer
and Campos-Ortega (Scheer and
Campos-Ortega, 1999). About 25% of embryos were affected by
dnSu(H) and NICD in these lines, respectively. Embryos were developed in fish
water containing methylene blue at 28.5°C and staged according to standard
protocols (Kimmel et al.,
1995). At least 30 embryos were observed for each time point,
except where noted.
In situ hybridization
In situ hybridization was performed at 67°C as described
(Jowett and Yan, 1996;
Phillips et al., 2001).
Immunofluorescence
Antibody staining was performed as described by Riley et al.
(Riley et al., 1999). Primary
antibodies: Pax2 (Covance, diluted 1:100), acetylated tubulin (Sigma T-6793,
diluted 1:100). Secondary antibodies: Alexa 546-conjugated goat anti-rabbit
IgG (Molecular Probes A-11010, diluted 1:50) or Alexa 488-conjugated goat
anti-mouse IgG (Molecular Probes A-11001, diluted 1:50).
Misexpression
The atoh1a plasmid was obtained from Reinhard Köster. To
misexpress atoh1a under the control of the cytomegalovirus promoter,
30-90 pg plasmid was injected into one-cell embryos. For RNA misexpression,
wild-type mRNA was synthesized in vitro using mMessage mMachine kit (Ambion).
A total of 60-80 pg mRNA was injected into one-cell embryos, or was
co-injected with atoh1a;atoh1b double MO.
Morpholinos
Morpholino oligomers (MOs) were obtained from Gene Tools, Inc. For most
experiments, 5 ng morpholino was injected into one-cell embryos. MOs for
dlx3b, dlx4b, fgf3, foxi1, pax2b and pax8 were described
previously (Solomon and Fritz,
2002; Mackereth et al.,
2005). Additional MO sequences are as follows: atoh1b MO
5'-TCATTGCTTGTGTAGAAATGCATAT-3'; atoh1a MO1
5'-TCTGTTGGTTTGTGCTTTTGGGAGG-3'; atoh1a MO2
5'-AAAGTTTGTGGCTATGGATACAGGG-3'; atoh1a MO3
5'-ATCCATTCTGTTGGTTTGTGCTTTT-3'. atoh1a MO3 was used for
most experiments. The phenotypes caused by injection of atoh1a and/or
atoh1b MOs affected 90-100% of embryos, except where noted.
SU5402 inhibitor treatment
SU5402 was dissolved in DMSO to prepare a 40 mmol/l stock solution. Embryos
were treated in their chorions with 50 µmol/l SU5402 (10-14 hpf), 80
µmol/l (12-18 hpf), or 100 µmol/l (18-24 hpf). Controls were incubated
in an equal concentration of DMSO to that of treated embryos. To terminate
treatment, embryos were washed several times and either allowed to develop
further or fixed and processed immediately.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Whitfield et al., 2002). We
designed three different MOs to block translation of atoh1a, all of
which affected hair cell development. While two of these MOs caused varying
degrees of non-specific cell death in the neural tube, the third was effective
at a dose that had no discernable toxicity and was therefore used for the
remainder of this study. Injection of atoh1a MO strongly impairs
formation of hair cells in the inner ear
(Fig. 2U). Tether cells, an
early-forming hair cell required for otolith localization
(Riley et al., 1997), were not
affected in atoh1a morphants, and otoliths formed normally
(Fig. 2G). Tether cells, named
for their precocious kinocilia, initially formed in pairs at both ends of the
nascent otic vesicle and later adopted the morphology of fully developed hair
cells by 22 hpf. Normally, later-forming hair cells begin to accumulate soon
after 24 hpf. However, later-forming hair cells were profoundly impaired in
all atoh1a morphants, as additional hair cells were not evident until
48 hpf (Fig. 2I,U, and data not
shown).
Adolf et al. (Adolf et al.,
2004) recently described a second zebrafish atonal
homolog, atoh1b, that we hypothesized might also play a role in hair
cell development. By contrast to atoh1a MO, injection of
atoh1b MO ablated tether cells in both the utricle and saccule
(Fig. 2B) in all specimens.
Later-forming hair cells were still produced, albeit more slowly than normal
(Fig. 2F,U). A single otolith
was produced but initially formed as an untethered mass due to the absence of
tether cells (Fig. 2D).
Otoliths eventually bound to utricular hair cell cilia after 30 hpf (not
shown).
Co-injection of atoh1a MO and atoh1b MO ablated all hair cells in the inner ear in >90% of specimens (Fig. 2J,U). This was confirmed using phalloidin to mark stereocilia and anti-actetylated tubulin staining of kinocilia (not shown). A single untethered otolith was produced (Fig. 2H), reflecting loss of tether cells. Hair cells did begin to form by 48 hpf in atoh1a;atoh1b double morphants (Fig. 2K,U), probably reflecting diminishing capacity of the MOs to knock down atoh1 function at later stages. Thus, atoh1 function is essential for hair cell formation in zebrafish, as in mouse. Moreover, the data support a model in which atoh1b preferentially regulates development of tether cells, whereas atoh1a regulates later-forming hair cells.
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; Sarrazin et al.,
2006) (B.B.R., unpublished).
Misexpression of atoh1a
To test whether the effects of atoh1 MOs on hair cell development
could be rescued, atoh1a;atoh1b double morphants were
co-injected with 80 pg of atoh1a mRNA. More than half of these
co-injected embryos produced tether cells, tethered otoliths and later-forming
hair cells (Fig. 2M-O),
indicating substantial rescue from the effects of the MOs. These data show
that loss of hair cells in atoh1 morphants is a specific consequence
of disrupting atoh1 function.
Injecting 80 pg of atoh1a mRNA (with or without MOs) did not lead
to formation of excess or ectopic hair cells. This is in contrast to mouse, in
which misexpression of atoh1 promotes formation of ectopic hair cells
in tissues immediately surrounding endogenous sensory epithelia
(Zheng and Gao, 2000;
Woods et al., 2004;
Izumikawa et al., 2005).
Because injected mRNA may not be stable enough to strongly affect later stages
of otic development, we injected zebrafish embryos with plasmid DNA to
misexpress atoh1a under the control of the powerful and ubiquitously
expressed cytomegalovirus promoter. Injection of 90 pg of atoh1a
plasmid caused axial truncation in up to 30% of embryos, whereas injection of
30 or 60 pg did not alter overall embryonic morphology (not shown). Embryos
injected with 60 or 90 pg of atoh1a plasmid often showed expanded
sensory patches at 24 hpf (Fig.
2Q). By 30 hpf, however, many supernumerary hair cells were lost,
whereas isolated Pax2-positive cells appeared sporadically in the subjacent
mesenchyme (Fig. 2R). The
latter are likely to be dying hair cells, as suggested by general elevation of
Acridine Orange staining (not shown). We showed in another study that dying
hair cells are often extruded from the otic vesicle to the underlying
mesenchyme (Kwak et al.,
2006). This also occurs in mind bomb (mib)
mutants, which form supernumerary hair cells that are later extruded as they
undergo apoptosis (Haddon et al.,
1999). It is possible that excess hair cells die because forced
expression of atoh1a bypasses vital processes required for hair cell
maintenance. We also cannot exclude the possibility of non-specific toxicity
associated with concentrated plasmid injection. In addition to changes in the
otic vesicle, about one-third of embryos injected with atoh1a plasmid
also formed ectopic Pax2a-positive cells in the surface ectoderm just anterior
or posterior to the otic vesicle. Double labeling with acetylated tubulin
antibody confirmed that some of these cells were hair cells
(Fig. 2S). Although ectopic
hair cells formed at the level of the lateral line, pax2a expression
indicated that these were not lateral line neuromasts. These data show that in
zebrafish, as in mouse, atoh1 misexpression can induce excess and
ectopic hair cells, but only in regions close to the endogenous hair cell
domains. This is consistent with findings that basic helix-loop-helix proteins
work combinatorially with other transcription factors, such as Hox and Pax
proteins, with regional expression that establishes restricted zones of
competence (Niwa et al., 2004)
(reviewed by Westerman et al.,
2003).
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Expression of atoh1b began much earlier, marking the medial edge of the preotic placode by 10.5 hpf (Fig. 7A,B). This pattern resolved into two discrete patches by 14 hpf, encompassing the future sensory epithelia (Fig. 3D). At this stage, expression of atoh1b overlapped with that of atoh1a, but atoh1b was expressed at a higher level (compare Fig. 3A,D). By 22 hpf, atoh1b expression diminished and marked only a subset of the atoh1a domain (Fig. 3E,F). These differences in temporal expression are consistent with the notion that atoh1b acts early in otic development, whereas atoh1a predominates during later development of sensory epithelia.
Autoregulation and crossregulation of atoh1 gene expression
Because proneural genes often regulate their own expression, we examined
expression of atoh1a and atoh1b in embryos knocked down for
either or both functions. In atoh1b morphants, preplacodal expression
of atoh1b was not altered (not shown). However, atoh1b
expression failed to become restricted to two sensory primordia in the otic
placode at 14 hpf (compare Fig.
3D,P). Expression of atoh1b ceased by 16 hpf in
atoh1b morphants (Fig.
3Q and data not shown), indicating that atoh1b is
required to maintain its own transcription. Interestingly, macular expression
of atoh1b returned after 24 hpf
(Fig. 3R).
atoh1a was not expressed in atoh1b morphants until around 20 hpf and was limited to the utricular (anterior) macula (Fig. 3M,N). By 30 hpf, atoh1b morphants showed atoh1a expression in both utricular and saccular maculae, although the level of expression was lower than normal (Fig. 3O). These data show that atoh1a requires atoh1b for expression in the otic placode but not in the otic vesicle after 20 hpf. Once activated, atoh1a could be responsible for reactivation of atoh1b expression after 24 hpf (Fig. 3R).
In atoh1a morphants, atoh1a and atoh1b were expressed normally to 20 hpf (Fig. 3G,J, and data not shown). By 22 hpf, atoh1a morphants began to express atoh1a at higher than normal levels (Fig. 3H,I). Conversely, atoh1b expression was nearly extinguished by 22 hpf and could not be detected after 24 hpf (Fig. 3K,L). These data show that atoh1a is necessary to maintain atoh1b expression after 22 hpf and that atoh1a limits its own expression.
In atoh1a;atoh1b double morphants, atoh1b was expressed in an expanded domain at 14 hpf but was not maintained in the ear after 16 hpf (Fig. 3V-X and data not shown). Expression of atoh1a could not be detected until 22 hpf, after which it was expressed at higher than normal levels (Fig. 3S-U). Sections showed that the epithelium had only a single layer of columnar cells that expressed high levels of atoh1a (Fig. 3U, inset).
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Involvement of atoh1 genes in Delta-Notch signaling
Proneural genes often limit their own expression by transcriptional
activation of Delta (Dl), which in turn stimulates Notch (N) and thereby
inhibits subsequent proneural gene expression
(Baker and Yu, 1997;
Parks et al., 1997). In
support of this, knocking down atoh1b strongly inhibited expression
of dlA and dlD in the ear at 14 hpf
(Fig. 4C,D, and data not
shown). Similarly, knocking down atoh1a diminished dlA and
dlD expression at 22 hpf (Fig.
4A,B, and data not shown). Thus, atoh1 genes are required
for normal activation of delta gene expression.
To further investigate the role of Dl-N feedback, we examined
atoh1 function in mind bomb (mib) mutants. The
mib gene encodes an E3 ubiquitin ligase essential for Dl-N signaling
(Itoh et al., 2003).
mib mutants produced an enlarged domain of both atoh1a and
atoh1b at 14 hpf, mimicking the failure to restrict expression seen
in atoh1b morphants (Fig.
4G,H). Because both atoh1 genes remain fully active in
mib mutants, delta gene expression is also greatly expanded
and all cells in the equivalence group complete differentiation as hair cells
(Haddon et al., 1999;
Riley et al., 1999)
(Fig. 4E,I). However, injection
of atoh1a MO and atoh1b MO into mib mutants fully
suppressed these latter defects, blocking delta gene expression and
ablating all hair cells in all specimens
(Fig. 4F,J). These data further
support a role for atoh1 genes as upstream activators of Dl-N
signaling, which normally acts to limit and refine atoh1 expression
and function.
To test the temporal requirements for the canonical N pathway, we used a
transgenic line to express a dominant-negative form of Su(H) [dnSu(H)] under
the control of hsp70 promoter
(Wettstein et al., 1997;
Shoji et al., 1998;
Latimer et al., 2005). This
promoter induces high-level transcription within 15 minutes following heat
shock, providing a pulse of protein accumulation lasting several hours
(Scheer et al., 2002). Heat
shock induction of dnSu(H) at 8 hpf did not alter atoh1b expression
or hair cell development (not shown). However, heat shock at 10 hpf caused the
initially broad domain of atoh1b to be maintained to at least 13.5
hpf, about 2 hours longer than normal (Fig.
5B). By 14.5 hpf, expression became restricted to two discrete
domains that were larger than normal (Fig.
5E). This domain restriction presumably reflects resumption of
Dl-N signaling as the pulse of dnSu(H) subsides. However, the enlarged domains
showed no further reduction after 14.5 hpf and went on to form supernumerary
hair cells (Fig. 5H). Heat
shock at 12 hpf (after equivalence-group restriction had already begun) also
resulted in maintenance of two large domains and production of excess hair
cells (Fig. 5C,F,I). Heat shock
at 14 hpf had little effect on atoh1b expression or hair cell
formation (not shown). These data show that equivalence group restriction can
still occur after 13.5 hpf, but then atoh1b expression stabilizes by
14.5 hpf regardless of domain size, defining an interval during which cell
fates are specified.
To test how N gain of function affects atoh1 gene expression (as
in Fig. 1), we used a heat
shock-inducible Gal4-UAS system to drive expression of N intracellular domain
(NICD) (Scheer and Campos-Ortega,
1999). In this system, heat shock induced sustained NICD
expression for at least 17 hours (Scheer
et al., 2002). Heat shock induction of NICD at 9 or 10 hpf did not
prevent induction of atoh1b in the preotic placode
(Fig. 6B). However,
atoh1b expression was lost by 12 hpf
(Fig. 6D). In addition,
atoh1a was never activated and no hair cells were produced (not
shown). Heat shock induction of NICD at 18 hpf also rapidly extinguished
atoh1 expression and blocked hair cell formation (not shown). We also
examined the effects of NICD in atoh1b morphants, which usually have
no functional equivalence group until 20 hpf, when atoh1a is first
expressed. In atoh1b morphants, activation of NICD at 18 hpf induced
atoh1a by 19 hpf, 1 hour earlier than without NICD
(Fig. 6E-G). Expression then
subsided by 20 hpf and no hair cells were produced
(Fig. 6H, and data not shown).
Thus, NICD initially stimulates, or at least does not block, upregulation of
atoh1 genes as the equivalence group forms but then rapidly
extinguishes atoh1 expression at all later stages.
In summary, the relationship between atoh1 function and the Dl-N pathway is consistent with all predictions of the fly ato paradigm (Fig. 1). Moreover, atoh1-dependent restriction of the equivalence group precedes fate specification by several hours. These findings strongly support a classic proneural mechanism of action for zebrafish atoh1 genes.
Regulation of atoh1b in preotic cells
Expression of pax8 is the earliest known marker of otic placode
induction (Pfeffer et al.,
1998). atoh1b is expressed in a subset of
pax8-expressing cells in the preotic placode
(Fig. 7A,B), raising the
possibility that pax8 is required for early activation of
atoh1b. Knocking down pax8 reduced the size of the preotic
domain of atoh1b (Fig.
7F), but the level of expression appeared normal. We next asked
whether factors that act upstream of or parallel to pax8 might also
regulate atoh1b. Induction of pax8 requires Foxi1
autonomously within the preplacodal ectoderm, as well as stimulation by Fgf3
and Fgf8 secreted from adjacent hindbrain tissue
(Phillips et al., 2001;
Maroon et al., 2002;
Leger and Brand, 2002;
Liu et al., 2003;
Solomon et al., 2003;
Hans et al., 2004). Knocking
down foxi1 caused severe reduction of atoh1b expression
(Fig. 7G). To test the role of
Fgf, embryos were treated with the Fgf signaling inhibitor SU5402. Induction
of atoh1b was blocked in embryos treated from 10-14 hpf (not shown).
When SU5402 was added beginning at 10.5 hpf, after the onset of
atoh1b expression, expression of atoh1b was lost in all
specimens by 12.5 hpf (Fig.
7D). Expression of atoh1a was also blocked
(Fig. 7I), consistent with a
requirement for atoh1b in atoh1a induction. Embryos
co-injected with fgf3 MO and fgf8 MO also did not express
atoh1 genes (not shown). Thus, Foxi1 and Fgf signaling are required
to initiate and maintain expression of atoh1b in the preotic placode,
and Pax8 is needed to produce a normal-sized domain.
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;
Liu et al., 2003;
Hans et al., 2004).
Accordingly, neither atoh1a nor atoh1b were expressed in
dlx3b;dlx4b morphants during placodal development
(Fig. 7E,J). Similarly,
b380 mutants, which are deleted for dlx3b and dlx4b
(Fritz et al., 1996), also
failed to express atoh1 genes in the otic placode (not shown). Later
in development, dlx3b;dlx4b morphants produced small otic
vesicles containing only anterior (utricular) sensory patches. Tether cells
did not form, consistent with loss of early atoh1b, but later hair
cells began to form after 24 hpf (not shown) in association with belated
expression of atoh1a (Fig.
7O). Dlx proteins could act directly on atoh1b
transcription or indirectly by regulating competence to respond properly to
Fgf after initial otic induction, as suggested by recent studies
(Hans et al., 2004;
Solomon et al., 2004).
Pax2 and Pax8 proteins maintain atoh1b
Pax8 normally cooperates with closely related proteins Pax2a and Pax2b to
maintain the otic placode (Hans et al.,
2004; Mackereth et al.,
2005). Knockdown of pax8 and pax2b in embryos
homozygous for a null mutation in pax2a (noi mutants)
(Lun and Brand, 1998) causes
progressive loss of otic tissue and no vesicles are produced. Accordingly such
embryos do not express atoh1a or atoh1b in the otic region
(not shown). Reducing the MO concentration by half allows the majority of
pax2a-pax2b-pax8-deficient embryos to produce small
otic vesicles. In 100% of these specimens, atoh1a was expressed at a
high level in a nearly normal number of cells at the anterior end of the otic
vesicle, whereas atoh1b expression was barely detectable in any
specimen (Fig. 7K,L). Partial
knockdown of pax8 and pax2b in wild-type embryos resulted in
a moderately diminished otic vesicle expressing normal levels of both
atoh1a and atoh1b (Fig.
7M,N), although atoh1b was typically expressed in only
one or two cells. These data show that full expression of atoh1b
requires Pax8 and Pax2 functions. By contrast, atoh1a expression is
not strictly dependent on Pax2 or Pax8 function.
Continuing requirements for Fgf
As the otic vesicle forms, fgf3 and fgf8 begin to be
expressed in domains encompassing the sensory epithelia
(Leger and Brand, 2002). To
test whether Fgf signaling regulates atoh1 expression after placode
formation, embryos were treated with SU5402 for various intervals at
successively later stages of development. Treatment from 12-18 hpf did not
affect atoh1b but reduced expression of atoh1a
(Fig. 8A-D). When embryos were
treated at 18 hpf for 1, 2, 4 or 6 hour intervals, expression of both
atoh1a and atoh1b were strongly reduced but not eliminated
(Fig. 8E-H). We hypothesized
that the period of SU5402 insensitivity of atoh1b from 12 to 18 hpf
reflects maintenance of atoh1b by autoregulation. Furthermore, as
atoh1a and atoh1b help maintain each other at later stages,
crossregulation could account for residual expression seen in SU5402-treated
embryos. In support of this hypothesis, atoh1b morphants failed to
express either atoh1a or atoh1b when treated with SU5402
from 18-22 hpf (Fig. 8J). We
next tested the effects of SU5402 on hair cell formation. In embryos treated
from 18-24 hpf, tether cells were produced normally (not shown). This was not
unexpected, because tether cells are already present in the otic vesicle at 18
hpf and hence their specification cannot be blocked by this treatment.
However, production of later-forming hair cells was strongly impaired during
the 6 hour period following removal of the inhibitor
(Fig. 8L,
Fig. 2V). Presumably the severe
reduction in atoh1 expression seen at 24 hpf delays resumption of
macular development. These data show that atoh1 expression and hair
cell development require ongoing Fgf signaling. This marks the first
identification of a signaling molecule required to both induce and maintain
atoh1 expression in the vertebrate inner ear.
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;
Kwak et al., 2006), was also
unaltered in atoh1a;atoh1b double morphants
(Fig. 9F). By contrast,
knockdown of both atoh1a and atoh1b strongly reduced the
level of pax2b expression (Fig.
9H). pax5 and pax2b are both required for normal
development and maintenance of hair cells
(Whitfield et al., 2002;
Kwak et al., 2006), but only
the latter was affected by atoh1 function. Thus, expression of
fgf genes and some downstream targets (pax5, atoh1a)
continue in the macular region despite disruption of atoh1 function
and the absence of a sensory epithelium. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fgf signaling is an essential upstream activator of atoh1
expression during both phases, although atoh1b becomes independent of
Fgf after 12 hpf. This could reflect the onset of atoh1b
autoregulation. A similar transition occurs with Drosophila ato,
which becomes autoregulatory as it initiates domain restriction and lateral
inhibition (Sun et al., 1998).
Fgf may facilitate the transition to autoregulation of atoh1b, in a
similar way to the role of Egfr and Map kinase activity in promoting
autoregulation of Drosophila ato during sensory organ development
(zur Lage et al., 2004).
Unlike atoh1b, maintenance of atoh1a remains heavily
dependent on Fgf but is not dependent on atoh1 function after 20 hpf.
Indeed, atoh1a;atoh1b morphants maintain higher than normal
expression of atoh1a. This is probably because fgf genes
continue to be expressed (Fig.
9) and promote atoh1a expression in the absence of
N-mediated feedback inhibition.
The overlapping yet distinct functions of zebrafish atoh1 genes
probably reflects evolutionary `subfunctionalization'
(Force et al., 1999).
Following a genome duplication thought to have occurred early in the teleost
lineage, duplicate copies of genes often diverge in regulation to subdivide
the ancestral function. Only atoh1b is required for development of
tether cells, which are analogous to primary neurons. Because such precocious
cell types are typical of anamniote embryos, this probably reflects an
ancestral atoh1 function. atoh1a has apparently lost
regulatory elements required to respond to the fgf-foxi1-pax and
dlx pathways involved in atoh1b induction. However, only
atoh1a is essential for later hair cells, which continue to form well
beyond embryonic development. This, too, is probably an ancestral
atoh1 function. Sensory epithelia continue to expand throughout life
in teleosts, suggesting ongoing recruitment of new cells into the equivalence
group. Fgf-dependent induction of atoh1a in adjacent cells might
account for such recruitment, a function similar to the role of Egfr and
ato in recruiting new sensory organ precursors in the
Drosophila chordotonal organs
(zur Lage et al., 1997). The
two Atoh1 proteins probably retain similar DNA-binding properties, however, as
misexpression of atoh1a can restore tether cell formation in
atoh1a;atoh1b double morphants
(Fig. 1M-O).
|
|
). Specific comparisons between zebrafish atoh1 genes
and Drosophila ato (Fig.
1) reveal striking parallels. More generally, various authors have
used four criteria to define proneural function
(Brunet and Ghysen, 1999;
Hassan and Bellen, 2000;
Westerman et al., 2003) that
can be applied to zebrafish atoh1 genes. First, proneural genes are
expressed before sensory fate specification. atoh1b is induced
broadly in the preotic placode at 10.5 hpf, whereas specification of tether
cells (stabilization of atoh1 expression) does not occur until 14
hpf. Second, proneural genes are subject to lateral inhibition (and the
related process of domain restriction) via N-mediated repression. Zebrafish
atoh1 genes, once induced, are readily repressed by N activity.
Moreover, both atoh1 genes facilitate their own repression by
autonomously activating delta expression. Third, proneural function
is necessary for producing the equivalence group for the entire sensory
structure. atoh1a;atoh1b morphants produce only a simple
epithelium lacking hair cells; and while support cell markers are not known in
zebrafish, it is important to note that the epithelium continues to express
atoh1a. As loss of atoh1 expression marks the first step in
support cell specification, these cannot be support cells. Fourth, proneural
function is sufficient to induce ectopic sensory development. Misexpression of
atoh1a induces ectopic hair cells, although only in limited regions
near the otic vesicle or endogenous sensory epithelia, as has been shown for
Atoh1 in mammals (Zheng and Gao,
2000; Woods et al.,
2004; Izumikawa et al.,
2005). Competence to respond appropriately to Atoh1 may require a
unique combination of additional factors. The zone of competence could be
influenced by pax2-5-8 genes, which are co-regulated with
atoh1 genes by Fgf signaling. Other signaling pathways have also been
implicated in this process. Misexpressing components of the Notch or Wnt
pathways in chick can also induce ectopic sensory patches, but only in
restricted regions near endogenous sensory patches
(Stevens et al., 2003;
Daudet and Lewis, 2005).
Combinatorial signaling and restricted zones of competence also influence the
functions of proneural genes in Drosophila
(Westerman et al., 2003;
Niwa et al., 2004). Thus,
while many additional details need to be resolved, zebrafish atoh1
genes meet all four criteria used to define proneural function.
Conserved mechanisms?
While mammals show no early phase of specification analogous to tether cell
development, and sensory epithelia develop only during a limited stage of
embryogenesis, some aspects of sensory development have been conserved. The
clearest example is the role of N signaling. Dll1 and Jag2
encode N ligands that regulate the balance of hair cells and support cells in
the mouse cochlea. Loss of Jag2 causes a modest increase in hair
cells (Lanford et al., 1999;
Kiernan et al., 2005a), as
does antisense knockdown of N1 in cochlear cultures
(Zine et al., 2000). Loss of
Dll1 causes a larger increase in hair cells
(Brooker et al., 2006), and
disrupting both Dll1 and Jag2 causes a dramatic increase in
hair cells and a modest decrease in support cells
(Kiernan et al., 2005a). The
number of support cells is greater than expected, because support cells
continue to divide for longer than normal, partially offsetting earlier
deficiencies. Although no phenotype comparable to zebrafish mib has
been described in mouse, the mouse data nevertheless support the lateral
inhibition model well. Residual support cell development probably reflects the
activity of another N ligand, Jag1. Jag1 is initially expressed
throughout the prospective sensory region and later becomes restricted to
support cells during differentiation. It has been proposed that Jag1 signaling
between support cells augments lateral inhibitory signals from hair cells
(Eddison et al., 2000).
Indeed, partial loss of Jag1 also leads to excess hair cell
production (Zine et al., 2000;
Kiernan et al., 2001).
However, conditional knockouts of Jag1 ablate much, although not all,
of the sensory epithelia (Brooker et al.,
2006; Kiernan et al.,
2006). This supports a model in which the function of Jag1 changes
with time, initially promoting the early inductive phase of N signaling and
later augmenting lateral inhibition. While the mechanistic basis for the shift
from inductive to repressive N signaling remains unknown, similar transitions
occur in the regulation of Drosophila ato and zebrafish
atoh1a (Baker and Yu,
1997) (Fig. 6F,H).
It is not known whether mouse also shows N-dependent restriction of the
initial equivalence group.
|
). Furthermore, Pirvola et al.
(Pirvola et al., 2002) have
proposed that Fgfs produced by inner hair cells in the Organ of Corti
stimulate differentiation of later-forming outer hair cells through activation
of Fgfr1.
A potential difference between mouse and zebrafish is the question of
whether mouse Atoh1 has proneural activity (reviewed by
Kelley, 2006). This is
especially evident when considering the mammalian cochlea, which is a highly
derived structure that differs in important ways from the more primitive
maculae and cristae. However, as summarized below, available data are complex
and can be considered inconclusive. Atoh1 is necessary for hair cell
differentiation and is sufficient for inducing ectopic hair cells
(Bermingham et al., 1999;
Zheng and Gao, 2000;
Woods et al., 2004;
Izumikawa et al., 2005).
Atoh1 is also subject to autoregulation
(Helms et al., 2000), which in
other species facilitates pattern refinement during lateral inhibition.
Unfortunately, a direct link between lateral inhibition and Atoh1 has
not been shown in mouse. Atoh1 is initially expressed in a broad
domain that spans the full depth of the epithelium, approximately four to five
cells thick (Bermingham et al.,
1999; Lanford et al.,
2000; Chen et al.,
2002; Woods et al.,
2004), but expression is not uniform and some cells appear to
express little or no Atoh1. These data do not distinguish whether
there is an earlier stage of low uniform Atoh1 expression followed by
rapid upregulation and pattern refinement or, alternatively, whether
Atoh1 marks only differentiating hair cells after fate specification.
Several groups have concluded that mouse Atoh1 lacks proneural
activity based in part on the observation that sensory regions in
Atoh1 knockout mice contain a single layer of cells that
morphologically resemble support cells
(Bermingham et al., 1999).
However, these cells express no definitive markers of mature support cells
(Woods et al., 2005). Early non-restricted expression of Jag1 occurs
normally, but later expression normally associated with support cells is lost.
Thus, support cell differentiation is disrupted, although it is not clear
whether the defect lies in specification or maintenance. Another early marker
of the sensory epithelium, p27kip1, normally precedes
Atoh1 in expression and continues to be expressed in the prosensory
region in Atoh1 mutants (Chen et
al., 2002). This has been interpreted to mean that cells of the
equivalence group are specified but fail to differentiate. However,
p27kip1 plays no role in fate specification, and there are
no independent indicators of when the equivalence group forms in mouse. While
expression p27kip1 is regulated partly by the same
inductive signals that specify the equivalence group
(Kiernan et al., 2006),
upregulation of fate-specifying gene(s) need not follow precisely the same
timecourse. Moreover, even if Atoh1 were necessary for prosensory
induction, loss of Atoh1 would not be expected to block any of the
initial transcriptional responses to inductive signals. Thus expression of
p27kip1 and Atoh1 in the absence of
Atoh1 function (Bermingham et al.,
1999; Chen et al.,
2002; Fritzsch et al.,
2005) could simply reflect ongoing parallel responses to common
upstream activators in cells that are otherwise blocked at an early stage.
Similarly, we have shown that several early markers of sensory epithelia in
zebrafish (atoh1a, pax5) are co-regulated by Fgfs and continue to be
expressed in atoh1a;atoh1b morphants
(Fig. 9). A similar situation
has been documented in Drosophila ato mutants, which produce no
photoreceptors in the eye but continue to coexpress genes normally preceding
formation of the prosensory equivalence group, including ato and the
N target gene hairy (Jarman et
al., 1995). In summary, gene expression and genetic studies in
mouse do not necessarily contradict the notion that Atoh1 might have
proneural activity, but key supportive data are also lacking. Resolving this
issue will require assessment of precisely when fate specification occurs
relative to expression of Atoh1 and p27kip1, how
these genes are co-regulated, and the epistatic relationships between the
various upstream factors, including Sox2, Jag1 and Fgf.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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