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First published online 13 February 2008
doi: 10.1242/dev.014142
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Mammalian Development Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Author for correspondence (e-mail:
ha3p{at}nih.gov)
Accepted 7 January 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Retina, Retinal pigment epithelium, Mitf red-eyed white, Mitf black-eyed white, Chx10-ocular retardation, Internal start codons
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sandelin et al., 2007).
Nevertheless, despite a wealth of sequence information for many species,
relatively little is known about the role and regulation of alternative
promoter use in vivo, both during development and in adulthood.
A prime example of a gene with multiple promoters is Mitf
(microphthalmia-associated transcription factor), which encodes a basic
helix-loop-helix leucine-zipper protein that is crucial for mammalian eye
development (Hodgkinson et al.,
1993; Nakayama et al.,
1998; Nguyen and Arnheiter,
2000). In mice and humans, the gene contains nine distinct
promoters, six of which are linked to different coding exons and three to
non-coding exons (Fig. 1)
(Hallsson et al., 2000;
Steingrimsson et al., 2004;
Hershey and Fisher, 2005;
Arnheiter et al., 2006;
Hallsson et al., 2007). Using
probes that do not distinguish between the corresponding isoforms, it has been
shown that Mitf is initially expressed in mice throughout the budding
optic vesicle and retained in the future retinal pigment epithelium (RPE), the
ciliary body and the iris, but is downregulated in the future retina and optic
stalk (Bora et al., 1998;
Nguyen and Arnheiter, 2000).
In Mitf mutants in which all isoforms are equally affected, the RPE
can develop as a laminated second retina
(Müller, 1950;
Bumsted and Barnstable, 2000;
Nguyen and Arnheiter, 2000),
and in mutants in which Mitf is abnormally upregulated in the retina,
the retina can develop as a single-layered RPE
(Rowan et al., 2004;
Horsford et al., 2005;
Bharti et al., 2006). From
these results, however, it remains unclear which isoforms are normally
expressed during RPE and retina formation and whether any of these isoforms
have selective roles or are entirely interchangeable.
The question of isoform-specific function and expression also touches on
the problem of whether distinct isoforms are co-regulated or regulated
separately. In fact, two lines of evidence argue in favor of co-regulation.
One is the observation, made in mice, that the combined genetic lack of the
eye transcription factors PAX2 and PAX6 is associated with a total absence of
all MITF protein. This finding is supported by the notion that the human
A-MITF promoter, which, like the mouse A-Mitf promoter, is located at the
5' end of the gene, is stimulated by PAX2/PAX6 in vitro, and hence might
serve as a control region for the entire MITF locus in the eye in
vivo (Bäumer et al.,
2003). The other is the recent observation that the paired-like
homeodomain transcription factor CHX10 (also known as VSX2 - Mouse Genome
Informatics), which is involved in the retinal downregulation of Mitf
(Liu et al., 1994;
Burmeister et al., 1996;
Rowan et al., 2004;
Horsford et al., 2005), may
stimulate a cluster of genes encoding microRNAs that serve to suppress
Mitf mRNAs by recognizing their common 3'UTRs
(Xu et al., 2007). Without
more-detailed information about the developmental expression pattern of each
Mitf isoform, however, an isoform-selective regulation cannot be
excluded a priori.
Here, we use a combination of tissue dissection, reverse transcriptase-PCR and isoform-specific antibodies to show that some Mitf isoforms are ubiquitously expressed throughout development in both ocular and extraocular tissues, that others are more restricted to the optic neuroepithelium, and others still are retained exclusively in the RPE. We then analyze two different mouse mutations associated with over- or underexpression of Mitf to assess the functional relevance of some of these isoforms in both retina and RPE. The results indicate that contrary to previous assumptions, the regulation of Mitf during eye development is not simply global, affecting all transcriptional isoforms indiscriminately, but in fact is isoform-selective. They also suggest that some isoforms are more crucial than others for early cell-fate decisions in the developing eye, and hence for the formation of a functional adult eye.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antibodies, immunohistochemistry, immunofluorescence and in situ hybridization
Pan-specific rabbit antibodies against MITF have been described
(Nakayama et al., 1998). An
MITF exon 1B1b-specific antibody was prepared by immunizing rabbits with a
synthetic peptide (SRILLRQQLMREQMQEQERR) and affinity purifying the resulting
serum using a corresponding peptide column. A mouse monoclonal antibody to the
C-terminus of MITF was prepared by standard procedures after immunizing mice
with a recombinant mouse MITF fragment corresponding to residues 298-419 (exon
9) tagged with GST. Clone 6A5 (IgG1, 
) was used as ascites fluid made
from ICR SCID mice. A rabbit anti-mouse-tyrosinase serum was a gift from Dr
Vincent Hearing (National Cancer Institute, National Institutes of Health,
Bethesda, MD). The following antibodies were obtained from commercial sources:
rabbit anti-PAX6 and mouse monoclonal anti-TUJ1 (Covance Research); rabbit
anti-cyclin D1 and rabbit anti-phosphohistone (Upstate Biotechnology); mouse
monoclonal against V5 tag (Invitrogen); sheep anti-N-terminus of CHX10
(Abcam); sheep anti-C-terminus of CHX10 (Exalpha Biologicals).
Immunolabeling was performed on 14-µm cryostat sections as described
previously (Nakayama et al.,
1998), except for the exon-specific anti-1B1b antibody, which
required antigen retrieval by boiling the sections for 30 minutes.
In situ hybridization was performed using a pan-specific Mitf
probe as described (Nakayama et al.,
1998), or a PCR-generated exon 1B1b-specific probe (primer
sequences available upon request).
Reverse transcription (RT) reactions and real-time PCR
RNA was isolated by two means. (1) RNA extractions using the RNeasy Mini
Kit (Qiagen). E9.5 and 10.5 eye primordia were manually dissected along with
the surrounding tissue. E11.5-P0 retinal tissue was separated manually from
the surrounding RPE/mesenchyme, except where indicated. cDNA was prepared
using the Superscript RT-PCR Kit (Invitrogen), using 1 µg of total RNA, and
cDNA corresponding to 25 ng of total template RNA was used for each PCR
reaction. (2) RNA was isolated using the Picopure RNA Extraction Kit
(Arcturus) from 100 cells that were microscopically selected after
trypsinization, using micromanipulator-controlled glass microinjection
pipettes. In these cases, the amount of cDNA used for amplification with two
rounds of PCR using nested primer pairs was estimated to correspond to
approximately 1-10 pg of template RNA.
Real-time PCR was performed using an ABI Prism 7000 real-time PCR machine
(Applied Biosystems). All RT-PCR products were sequenced at least once to
confirm primer specificity. In the eye, sequencing provided no evidence for
alternative splicing events linking exon 1A with exon 1J1b, nor exon 1J1b with
exon 1C1b, as reported for cultured cell lines
(Hershey and Fisher, 2005).
Therefore, exons 1J and 1C are here considered as single exons. Primer sets
used for RT-PCR and real-time PCR are available upon request.
For Fig. 3B, eyes from 20 E11.5 and 12 E15.5 wild-type embryos were manually dissected to give separate RPE/mesenchymal and retinal fractions. RNA was isolated using the RNeasy Mini Kit. The following procedure was used to generate standard curves and assay the test samples (Fig. 3B). First, isoform-specific cDNAs were generated by PCR using isoform-specific forward primers and a common reverse primer in exon 1B1b. Second, the cDNAs were quantitated spectrophotometrically and quantitation was confirmed by agarose gel electrophoresis. To generate standard curves, appropriate concentrations of the quantitated cDNAs were diluted into cDNA prepared from hearts of Mitfmi-rw/mi-rw animals. The rw cDNA lacks exon 1B corresponding to the common reverse primer but mimics the cDNA complexity of the test samples, thereby providing an amplification environment for the standards that is similar to that provided by the test samples. Real-time PCR of the cDNA standards was performed with exon-specific forward primers and a common reverse primer in exon 1B1b. Standard curves were generated for each isoform, and separately for each repeat assay performed with the test sample. Real-time PCR of the test samples was performed (four independent assays, each in triplicate), using appropriate primers. Absolute amounts of cDNAs were determined using the appropriate isoform-specific standard curves. The calculated amounts represent the levels of the respective isoforms in cDNA prepared from 25 ng of total RNA and confirmed as giving equal amplification of Gapdh and β-actin cDNAs. Considering that mRNAs represent approximately 3% of total RNA, and RT efficiency is approximately 10%, the isoform-specific levels shown in Fig. 3B correspond to approximately 75 pg of mRNA.
For Fig. 4, 14 whole eyes each from E12.5 wild-type and Mitfmi-rw/mi-rw embryos, and from P0 wild-type and Mitfmi-rw/mi-rw mice were used. RNA isolation, RT reactions, and real-time PCR were as above except that: (1) single-exon-specific primers were used; (2) the measurements were repeated three times, each time in triplicate; and (3) the results are given as the ratio between the amounts measured in Mitfmi-rw/mi-rw and in wild-type cDNAs. This means that unlike for Fig. 3B, there was no need for isoform-specific standard curves. However, to correct for variations between samples, wild-type and mutant results were first normalized to the internal β-actin and Gapdh levels.
Molecular characterization of the Mitfmi-rw mutation, constructs, in vitro mutagenesis and reporter assays
The Mitfmi-rw mutation was characterized by PCR and
Southern blots (see Fig. S1 in the supplementary material). The various
Mitf isoforms were cloned and their activities assayed in vitro (see
Fig. S2 in the supplementary material).
Chromatin immunoprecipitation (ChIP) assays
For details of ChIP assays using anti-CHX10 antibodies and amplification of
Mitf promoter DNA see Figs S3, S4 in the supplementary material.
Primer sequences are available upon request.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Nguyen and Arnheiter, 2000).
To analyze the isoform composition of this pan-specific signal, we first used
an in situ hybridization probe specific for exon 1B1b, which is common to all
isoforms except M-Mitf (Fig.
1), a major component of neural crest-derived choroidal and iris
melanocytes. As shown in Fig.
2A, exon 1B1b is expressed in the optic vesicle at E9.5, is
downregulated in the distal optic vesicle at E10.0, and is then concentrated
in the developing RPE as previously reported for a single time point
(Amae et al., 1998). As
expected, no labeling was seen in choroidal and iris melanocytes, which start
to accumulate at around E11.5 on the proximal side of the RPE and normally are
labeled with pan-specific probes (Nakayama
et al., 1998). Also, no labeling was seen on sections from
Mitfmi-rw (Mitf red-eyed white) embryos
(Fig. 2A, right-most panel),
which carry a genomic deletion of exon 1B
(Hallsson et al., 2000) but
express Mitf, as detectable with pan-specific probes (see below,
Fig. 5F).
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In immunohistochemical assays (Fig.
2C), both exon 1B1b- and pan-specific rabbit antibodies gave
overlapping expression patterns in the neuroepithelial parts of the developing
eye, starting from E9.5, when MITF is expressed in the entire optic vesicle,
through E17.5 and beyond (not shown), when it is expressed predominantly in
the RPE. Neural crest-derived choroidal melanocytes, however, were only
labeled with the pan-specific antibody (arrows in
Fig. 2C, panel labeled wt
E15.5). Both exon 1B1b- and pan-specific antibodies gave a low-level labeling
in the retina and surrounding mesenchyme when compared with equivalently
treated sections from Mitfmi-rw/mi-rw embryos, which lack
exon 1B (Hallsson et al.,
2000), or with Mitfmi-vga-9/mi-vga-9 embryos,
which lack MITF entirely (Hodgkinson et
al., 1993; Nakayama et al.,
1998) (Fig. 2C,
panels labeled Mitfmi-rw/mi-rw or
Mitfmi-vga-9/mi-vga-9). This result is consistent with the
observation presented in Fig.
2B, showing that E12.5 retinas continue to express at least some
isoform(s) of MITF.
Expression of promoter-specific Mitf isoforms
To determine the expression profile of individual isoforms, we exclusively
used RT-PCR, primarily because it provides the sensitivity to detect
low-abundance isoforms. For the early time points (E9.5-10.5), RNA was
obtained from whole-eye primordia including the surrounding tissue, and for
the later time points (E11.5-P0), separately from RPE/choroidal and retinal
fractions. cDNAs were amplified using either the respective 5'
exon-specific forward primers in combination with a reverse primer in exon
1B1b, or, in the case of M-Mitf, a reverse primer in exon 2A. Pan-specific
primers corresponded to exons 2A and 4. The number of PCR cycles was adapted
for each primer pair to give amplification in the linear range. As shown in
Fig. 3A, the first signal was
seen at E9.5 with pan-specific primers and weakly with A- and J-Mitf-specific
primers, whereas H- and D-Mitf-specific amplification gave the first signals
at E10.5. No other isoforms were detected at these early stages.
During the subsequent developmental time points, the different isoforms
could be grouped into four distinct expression patterns
(Fig. 3A). First, throughout
the period from E11.5 to P0, A- and J-Mitf showed similar expression profiles,
both in the retina and the RPE/choroid. Second, H-Mitf was preferentially
found in the RPE/choroid but was present at low levels in the retina as well,
particularly after E15.5. Third, D- and M-Mitf were found only in the
RPE/choroid and not the retina. Fourth, C-, MC-, E- and B-Mitf were
undetectable, or only barely detectable, throughout development in either
retina or RPE/choroid (not shown). In support of these results, from E11.5 to
P0, pan-specific amplification showed higher expression levels in RPE/choroid
compared with retina. Additional assays indicated that the
Mitf-related genes Tfe3, Tfeb and Tfec (also known
as Tcfe3, Tcfeb and Tcfec, respectively - Mouse Genome
Informatics) (Hallsson et al.,
2007), were also expressed in the developing eye, and
amplification for Chx10, which in the eye is retina-specific
(Liu et al., 1994), gave a
signal only in the retinal fraction. This latter finding, together with the
fact that D- and M-Mitf were only seen in the RPE/choroidal fraction,
indicates that efficient tissue separation had been achieved.
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). In postnatal eyes of such mice, M-Mitf mRNAs are
missing, full-length exon 1B1b-containing mRNAs are reduced, and skipping of
common downstream exons is increased
(Yajima et al., 1999). At
E11.5 through E17.5, however, we found no differences between
Mitfmi-bw and wild-type eyes in Mitf expression,
except that M-Mitf was lacking in the mutant (not shown). Hence, the use of
separately trypsinized RPE/mesenchymal and retinal fractions of E15.5
Mitfmi-bw/mi-bw allowed us to select microscopically small
populations (100 cells) of pure mesenchymal, RPE or retinal cells (for details
see Materials and methods). The respective cDNAs were amplified in two rounds
of PCR using nested primer sets. This analysis confirmed that A-Mitf is
ubiquitous and showed that H- and D-Mitf are not only present but in fact
enriched in the RPE, compared with retina and mesenchyme. No signals were
found for the low-abundance J-Mitf and, as expected, for M-Mitf
(Fig. 3B, lanes 1-3). The
results are consistent with those obtained from eye tissues dissected using
standard techniques (Fig. 3B,
lanes 4-6), where A-Mitf and low levels of J-Mitf were found in all fractions
(lanes 4-6), H- and D-Mitf were enriched in the fraction that contains RPE and
mesenchyme (lane 5), and M-Mitf was absent from all fractions.
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Genomic deletion of Mitf exons 1H, 1D and 1B leads to increased expression of novel mRNA and protein isoforms
To address the question of whether the major RPE isoforms, H- and D-Mitf,
are functionally relevant, we analyzed mice homozygous for the
Mitfmi-rw (Mitf red-eyed white, for short,
rw) allele which is characterized by a genomic deletion that
encompasses the 1H-, 1D- and 1B-exons
(Steingrimsson et al., 1994;
Hallsson et al., 2000). These
mice retain a patch of pigmented coat on the head and occasionally on the
belly and have eyes, the sizes of which can vary both between individuals and
between the left and right side of the same individual (example shown in
Fig. 4A, left side). We first
determined the exact boundaries of the deletion and found that it comprises a
stretch of 86,346 bp starting downstream of exon 1E and ending upstream of
exon 1M (Fig. 4A and see Fig.
S1 in the supplementary material). The deletion has the consequence that the
upstream exons, whether coding or not, splice into the next available
downstream exon, exon 2A, rather than the normal exon 1B1b
(Fig. 4A). Consequently, if
translation initiates at the normal start codons in exon 1A, 1C and 1MC, no
open reading frames are maintained beyond exon 2 (marked by red dotted lines
in Fig. 4A). Novel open reading
frames, however, are generated within the normally non-coding exons 1J and 1E
(blue lines in Fig. 4A), but
the potential AUG start codons do not conform to Kozak rules for efficient
translation initiation (Kozak,
1984; Kozak,
1986).
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1.0. Using primers in the specific upstream exons and exon 4, we further
confirmed that the overexpressed upstream exons were indeed part of spliced
RNAs (Fig. 4D). Note, however,
that even though the exon 1E-specific forward and reverse primers gave a
visible band in wild type (Fig.
4B), no correctly sized band was seen in wild type with primers in
exons 1E and 4 (Fig. 4D). This
confirms the finding mentioned above that spliced transcripts containing exon
1E are below the detection threshold in the developing eye.
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Deletion of Mitf exons 1H, 1D and 1B interferes with normal eye development but allows for partial pigmentation
To determine the in vivo consequence of the rw deletion, we
analyzed the development of rw eyes in detail. As shown in
Fig. 5, in situ hybridization
with a pan-Mitf-specific probe indicated that Mitf mRNAs are
indeed expressed at the expected locations in the developing rw eyes.
At E12.5, however, their levels were reduced compared with wild type, but at
E17.5, they were close to normal (Fig.
5, compare A with B, E with F). Immunohistochemistry using
pan-specific antibodies indicated a reduced signal in rw at both
ages, which is likely to reflect a reduced translational efficiency of the
rw mRNAs compared with wild-type RNAs
(Fig. 5, compare C with D, G
with H). Despite the low MITF protein levels, however, a clear signal for
tyrosinase was found in rw RPE, particularly at E17.5
(Fig. 5I-L), in contrast to
developing eyes from Mitf-null mutants, which lack tyrosinase
expression (Nakayama et al.,
1998). Consistent with this result, pigmentation, although at low
levels, could be found in the rw peripheral RPE and iris
(Fig. 5M,N) as well as in the
neuroepithelial part of the adult iris
(Fig. 5O,P). Nevertheless, RPE
development was perturbed, either because the overall MITF protein levels were
too low or because full-length proteins containing the normal N-terminal
sequences were missing. Much as in developing eyes of other Mitf
mutants (Müller, 1950;
Bumsted and Barnstable, 2000;
Nguyen and Arnheiter, 2000),
the dorsal RPE became thick (Fig.
5Q,R), retained high-level PAX6 expression
(Fig. 5Q-T), and eventually
transdifferentiated into a laminated second retina (not shown). In some eyes,
however, the RPE abnormalities were characterized not by a general thickening
but by foldings (Fig. 5S,T), a
much milder abnormality than seen in other rw eyes. The results
suggest that the lack of H- and D-MITF and exon 1B leads to RPE abnormalities
of variable severity, but that the novel MITF protein isoforms are still
capable of inducing pigmentation of the ciliary margin and the iris.
Lack of CHX10, a negative regulator of Mitf, leads to an increase in H- and D-Mitf in the retina
The downregulation of Mitf during retinal development involves a
pathway that is initiated by extracellular signals emanating from the surface
ectoderm, notably FGF1/2, and includes the paired-like homeodomain
transcription factor CHX10 that negatively regulates Mitf expression
in the retina (schematically indicated in
Fig. 6A). As previously shown
in Chx10orJ/orJ (Chx10-ocular retardation) mutant
embryos, which lack functional copies of Chx10, the future retina is
hypoplastic (Konyukhov and Sazhina,
1966; Burmeister et al.,
1996; Green et al.,
2003) and Mitf mRNA and protein levels are increased
(Rowan et al., 2004;
Horsford et al., 2005). To
confirm that the mutant retina hypoproliferates, we used phosphohistone H3
labeling to mark the cells at the G2-M transition. As expected, the thinner
Chx10or-J/or-J retina showed substantially fewer labeled
cells (Fig. 6B,C). As shown in
Fig. 6D,E, this phenotype is
correlated with the retinal retention of Mitf RNAs containing exon
1B1b. To dissect the isoform composition of the upregulated RNA, we used
limiting cycle RT-PCR of RNA extracted from wild-type and Chx10
mutant eyes at E13.5. The results (Fig.
6F) revealed a substantial increase in H- and D-Mitf, but a barely
perceptible increase in A- and J-Mitf. Theoretically, a specific retinal
upregulation of A- and J-Mitf might have been partially masked by the
ubiquitous expression of these isoforms outside the retina. This is unlikely,
though, because retinal changes in the expression of cyclin D1, which is also
expressed outside the retina, could easily be detected in the whole-eye
extracts, and A- and J-Mitf continued to be expressed throughout development
in the Chx10-positive wild-type retina (see
Fig. 2).
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Deletion of Mitf exons 1H, 1D and 1B corrects the retinal hypoproliferation phenotype in Chx10 mutant embryos
The above results prompted us to determine whether the lack of H- and
D-MITF and exon 1B, as seen in rw embryos, might be sufficient to
correct the Chx10-mediated retinal hypoproliferation phenotype. Such
a correction has thus far only been described with dominant-negative
Mitf mutations that equally affect all isoforms
(Konyukhov and Sazhina, 1966;
Horsford et al., 2005;
Bharti et al., 2006).
Therefore, we intercrossed Chx10orJ/orJ mice with
Mitfmi-rw/mi-rw mice to generate double homozygotes.
Staining for cyclin D1 in conjunction with PAX6 and the neuronal marker TUJ1
[β-3-tubulin (TUBB3)] indicated that, as expected, eyes from
Mitfmi-rw newborns
(Fig. 7A,E,I) had a retinal
thickness and lamination similar to those of wild type
(Fig. 7D,H,L), whereas eyes
from Chx10orJ newborns showed the severe retinal
hypoproliferation and retina-to-RPE transdifferentiation described previously
(Burmeister et al., 1996;
Rowan et al., 2004;
Horsford et al., 2005)
(Fig. 7B,F,J). In the double
Mitfmi-rw;Chx10orJ homozygotes, however, cyclin
D1 staining and retinal thickness were nearly normal, although retinal
lamination was still abnormal (Fig.
7C,G,K). This result indicates that in Chx10 mutants,
Mitf upregulation, once deprived of the contribution of H- and D-Mitf
and exon 1B, no longer leads to retinal hypoproliferation. This is consistent
with earlier observations showing that dominant-negative Mitf
mutations partially correct the Chx10 mutant phenotype
(Konyukhov and Sazhina, 1966;
Horsford et al., 2005).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fact that upstream Mitf regulatory regions contain distinct
transcription-factor-binding motifs that are conserved across mammalian and
avian species (Hallsson et al.,
2007) argues strongly for a transcriptional control of the
different Mitf isoforms. For instance, the ubiquitously active A-Mitf
promoter has potential binding sites for transcription factors with widespread
expression patterns such as MYC/MAX, STAT and SP1
(Hallsson et al., 2007). By
contrast, the H- and D-Mitf promoters have potential binding sites for the
retina-specific transcription factor CHX10
(Hallsson et al., 2007) (this
paper), precisely the factor involved in the retinal downregulation of these
two promoters. In fact, ChIP assays clearly indicate that at least some of
these sites are occupied by CHX10 in vivo, suggesting a direct regulation of
the respective Mitf promoters by this transcription factor.
|
). These binding sites, however, are not well conserved across
mammalian and avian species. Hence, it is possible that even in
Pax2/Pax6 double knock-out mice, the ubiquitously expressed A- and
J-MITF, which are undetectable by immunofluorescence on histological sections
(data not shown), might continue to be present, and only the major and
immunofluorescently detectable H- and D-MITF are absent. A different,
post-transcriptional co-regulatory mechanism is suggested by the conservation,
in the 3'UTR, of potential recognition sites for microRNAs, some of
which are thought to be controlled by Chx10
(Hallsson et al., 2007;
Xu et al., 2007). In fact,
microRNAs are excellent candidates to contribute to the retinal downregulation
of all Mitf mRNAs that share the same 3'UTR, but the
contribution of these microRNAs is likely to be small and might not exceed the
minor effects that Chx10 has, for instance, on the levels of A- and
J-Mitf.
The fact that each of the nine promoters is linked to a unique exon also
leads to the important question of whether multiple promoters exist solely to
provide the correct levels of MITF protein at each developmental time point
and location, or whether they exist to generate mRNAs or proteins with
isoform-specific functions. Indeed, some evidence suggests that sequence
differences at the MITF N-terminus can influence the proteins' activities. For
instance, ectopic expression of ascidian Mitf, the N-terminal
sequence of which resembles A-MITF, induces additional pigment cells in
ascidian larvae, as does mouse A-MITF, but not mouse H- or M-MITF
(Yajima et al., 2003).
Furthermore, A-, MC- and E-MITF regulate distinct sets of target genes in
Mitf-null mutant mast cells
(Shahlaee et al., 2007). There
is also evidence, however, that expression levels are more important than
specific sequences in determining MITF activities. For instance, regardless of
whether A-MITF or M-MITF was expressed in quail neuroepithelial cells,
pigmented colonies arose that consisted of RPE-like epithelial cells, in which
MITF was low, and of neural crest-like dendritic cells, in which MITF was high
(Planque et al., 2004). Our
findings with the natural Mitfmi-rw deletion suggest, in
fact, that low levels of N-terminally truncated MITF proteins are capable of
inducing pigmentation in the ciliary margin and the neuroepithelial portions
of the iris, whereas in the complete absence of Mitf no such
pigmentation is ever seen (Hodgkinson et
al., 1998; Nakayama et al.,
1998). This finding is consistent with the fact that N-terminally
truncated MITFs retain activity on promoters of pigmentation genes, including
the promoter of tyrosinase, which encodes the rate-limiting enzyme for melanin
synthesis (see Fig. S2 in the supplementary material). On the other hand, the
dorsal RPE is still abnormal in Mitfmi-rw embryos. It is
conceivable, therefore, that full RPE development, in contrast to mere
pigmentation, depends on the presence of the upstream coding exons, perhaps on
exon 1B1b in particular. This exon contains a glutamine-rich domain, thought
to be involved in protein-protein interactions, that is conserved in
vertebrates and flies (Hallsson et al.,
2007) and is reminiscent of similar domains in other transcription
factors such as CREB and SP1. That cell differentiation does not require this
domain, however, is evident from the fact that M-MITF is sufficient to induce
pigmentation upon experimental expression in cultured cells
(Bejar et al., 2003), and that
Mitfmi-rw mice show RPE and iris pigmentation as well as
some pigmented spots in the coat. In any event, a definitive answer to the
question of why there are distinct promoters must await results from targeted
promoter/exon knockouts and isoform-specific rescue experiments.
In addition to highlighting the importance of alternative promoter use, our
study of the Mitfmi-rw deletion also draws attention to
alternative translation initiation. Alternative translation start sites have
been observed in several genes and, much as with alternative promoter use, can
contribute to functional diversity in wild type and alter disease in mutants
(Sandelin et al., 2007;
Scheper et al., 2007;
Zhang et al., 2007). The fact
that in the rw mutant mice, translation can be initiated from
downstream start codons, suggests that these codons might be utilized for
initiation even in the context of a wild-type sequence, although at low
efficiency. Whether this is biologically relevant, however, remains to be
explored.
Finally, the fact that the Mitfmi-rw deletion is
associated with random bilateral asymmetries in eye development merits special
consideration. The fact that genetically identical cells, even if exposed to
the same environment, can show substantial variations in phenotypic
manifestations is thought to result from `stochastic' variations in gene
expression that are likely to be due to variant epigenetic chromatin states
(for a review, see Kaern et al.,
2005). It is reasonable to assume that high levels of gene
expression will generally buffer cells against random fluctuations in their
molecular composition and so ensure a normal embryonic development. If,
however, only threshold levels of gene expression are achieved, as is the case
for Mitf in rw mice with their abnormal Mitf
transcripts, stochastic fluctuations in epigenetic states might lead to major
phenotypic differences between individual cells. Given that epigenetic states
can be passed on through cell divisions, cellular variations might lead to
tissue variations and so to differences between organs that are normally
bilaterally symmetrical. Hence, our study of Mitf isoforms not only
touches on the complexities by which a single gene controls organogenesis, but
also on the intriguing question of how bilateral symmetries are being
generated and maintained.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/6/1169/DC1
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ACKNOWLEDGMENTS |
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