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J Biol Chem, Vol. 274, Issue 27, 19254-19260, July 2, 1999
From the To elucidate the regulatory mechanisms underlying
lens development, we searched for members of the large Maf family,
which are expressed in the mouse lens, and found three, c-Maf, MafB, and Nrl. Of these, the earliest factor expressed in the lens was c-Maf.
The expression of c-Maf was most prominent in lens fiber cells and
persisted throughout lens development. To examine the functional
contribution of c-Maf to lens development, we isolated genomic clones
encompassing the murine c-maf gene and carried out its
targeted disruption. Insertion of the Lens development commences in the 9.5-day-old (e9.5) mouse embryo
by invagination of the lens placode to form lens pits on either side of
the prospective forebrain (1, 2). Subsequently at e10.5, the lens pit
forms a lens vesicle, where embryonic ectodermal cells differentiate
into primary lens fiber cells. By e13.0, the primary posterior lens
fiber cells grow into the lumen to eventually fill the lens vesicle.
The anterior cells of the vesicle become epithelial cells and
constitute the lens germinal epithelium; secondary fiber cells then
differentiate from the epithelial cells after this stage. This
arrangement persists throughout the lifetime of the animal, as new lens
fibers are continuously regenerated (3).
Differentiation of the lens involves biosynthesis of a group of fibrous
lens-specific proteins called crystallins, which constitute 80-90% of
the soluble protein of the lens (4-6). The regulation of the
crystallin genes has been characterized extensively (7-10), and an
enhancer for the chicken Recently a new transcription factor, L-Maf, which can interact with the
Transcripts encoding other members of the Maf family in lens tissues
have been identified (Ref.
20-22)2 Nrl mRNA was
detected in embryonic mouse lens (20), whereas c-Maf and MafB mRNAs
were found in both embryonic and adult rat lens (21, 22), suggesting
that each of these individual large Maf proteins might play distinct
roles during lens development. Thus the vertebrate lens could provide
an excellent model system for dissecting both the individual as well as
complementary functional roles of large Maf family transcription factors.
To elucidate the functional contributions of Maf family factors to lens
development, we set out to identify the large Maf factors specifically
expressed in the mouse lens. We found that three large Maf family
proteins, c-Maf, MafB, and Nrl, are expressed in the embryonic and
adult mouse lens. We therefore documented the expression profile of
c-Maf and MafB mRNAs and also performed targeted disruption of the
c-maf gene in embryonic stem (ES) cells to generate
c-maf germ line mutant mice. Targeted mutation of the
c-maf gene results in perinatal lethality in homozygous
mutant animals, permitting us to examine the earliest stages of lens development in the embryo. These results demonstrate that c-Maf is
essential for normal lens development and that its function cannot be
complemented by other large Maf proteins.
Display of RNA Transcripts--
Total RNAs were prepared
from the lenses of e12.5- e18.5 embryos and adult mice. Degenerate
sense
(5'-GAGGGATCCATGGA(A/G)TA(C/T)GTIAA(C/T)GA(C/T)TT(C/T)GA) and antisense
(5'-GAGGAATTCGC(A/G)TAICCIC(G/T)(A/G)TT(C/T)TT) oligonucleotide primers containing engineered BamHI
and EcoRI recognition sites (underlined), respectively, were
synthesized. These primers correspond to peptide sequences MEYVNDFD and
KNRGYA, respectively, which are conserved between c-Maf, MafB, Nrl, and L-Maf. Coupled reverse transcriptase-polymerase chain reaction (RT-PCR)
was performed under the following conditions: annealing temperature of
50 °C and elongation temperature of 72 °C for 45 cycles.
In Situ Hybridization--
For whole mount in situ
hybridization analysis, e9.5-e11.5 embryos were dissected in
phosphate-buffered saline and fixed overnight in 4% paraformaldehyde
plus 2 mM EGTA in phosphate-buffered saline at 4 °C. The
embryos were then treated with proteinase K for at least 10 min
(depending on the embryo stage). After postfixation, the embryos were
prehybridized and then hybridized with RNA probes (23). The embryos
were subsequently incubated with alkaline phosphatase-conjugated
anti-digoxigenin antibody (Roche Molecular Biochemicals). Hybridization
signals were visualized using nitroblue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate as chromogen.
For cellular resolution in situ analysis, e9.5-e11.5 embryos
were fixed in 4% paraformaldehyde in phosphate-buffered saline. Embryonic lens tissue was cut into 16-20-µm frozen sections.
In situ hybridization was performed as described previously
(24). After hybridization, the sections were processed for
immunocytochemistry with anti-digoxigenin antibody, as described above.
To generate RNA probes for in situ hybridization of whole
mount and sectioned embryos, two ORF regions ( Southern Blot Hybridization Analysis--
High molecular weight
genomic DNA was extracted from P19 embryonic carcinoma cells following
standard procedures (25). Genomic DNA was digested with restriction
enzymes, electrophoretically separated, and then transferred to a nylon
membrane. The genomic fragment corresponding to 5'-Rapid Amplification of the cDNA End (5'-RACE)
Analysis--
An adapter oligomer-ligated cDNA library was
constructed using poly(A)+ RNA from e12.5 embryos and
Marathon cDNA amplification kit
(CLONTECH). Antisense primers complementary to the
5'-untranslated region were synthesized. Primer 1 (5'-TCCGCTGCGCGCTTTGCATAAGG-3') and primer 2 (5'-CCGTGCAAAGTGCAAGACCGAGGTGC-3') correspond to Targeted Disruption of c-maf--
c-maf genomic
clones were isolated from a 129/SVJ genomic library (Stratagene) using
a partial mouse c-Maf cDNA as probe. To construct the
gene-targeting vector, both the neomycin phosphotransferase (neo) and lacZ genes were inserted into the
c-maf ORF region between the MscI and
XhoI sites. The diphtheria toxin A (DT-A) gene (provided by
Dr. M. Taketo, University of Tokyo) was inserted at the end of the 3'
short arm of the targeting vector for negative selection. Targeted ES
cells were identified by PCR screening, as described previously
(26).
Independent clones, which had undergone homologous recombination at the
c-maf locus, were isolated, and their genotypes were verified by
Southern blot hybridization analyses using neo and c-maf probes. These clones were then injected into C57BL/6J
blastocysts. Offspring were genotyped by PCR and Southern blotting. To
distinguish between the wild type and gene-targeted
c-maf alleles by PCR, c-maf sense (W5':
5'-CTGCCGCTTCAAGACCCTCGACT-3') and neo sense (N5':
5'-CAGTCATAGCCGAATAGCCTCTCCACCCAA-3') primers were used with a single
c-maf antisense primer (W3':
5'-GGTCCCAGTCCCTCTATCTGTGCTCCTTCC-3'). The PCR profile used was
98 °C, 20 s and 68 °C, 5 min for 32 cycles in the presence
of 2.5 mM MgCl2. Southern blot analysis was
performed using genomic DNA digested with EcoRI and
NcoI-XhoI restriction endonucleases. To detect
the wild type and mutant alleles, the c-maf ORF region
(c-Maf probe; for detecting the 1.0 kilobase pair band) and a
neo gene fragment (neo probe; for 14.5 kilobase pairs) were
used, respectively. For LacZ staining, cryo sections were stained with
5-bromo-4-chloro-3-indolyl- Immunohistochemistry--
Embryos were fixed in ice-cold 10%
neutral formalin in phosphate-buffered saline for 2 h, dehydrated
with ethanol, embedded in paraffin, and sectioned at a 3-µm
thickness. The sections were dewaxed and incubated for 20 min with
anti- RT-PCR Analysis of Crystallin Gene Expression--
The heads of
e16.5 embryos were dissected, and total RNA was extracted individually.
After genotyping, 1 µg of the total RNA was used for cDNA
synthesis using Super Script IITM reverse transcriptase
(Life Technologies, Inc.) and random hexamer primers. Primer sets and
PCR conditions were as described previously (28).
Expression of Large Maf Family Factors in the Mouse Lens--
To
identify the large Maf family members that are expressed in the mouse
lens, we performed RT-PCR display analysis using degenerate
oligonucleotides encoding amino acid sequences that were conserved
among the large Maf factors. From both embryonic and adult lens cells,
seven PCR products were observed (data not shown). All seven PCR bands
were subcloned, and their sequences were determined by examining at
least five independent clones corresponding to each band. From the
sequence analysis, three of the bands were amplified from c-Maf
cDNA, and interestingly, all three c-Maf amplicons were
substantially smaller than predicted. We found that each of them was
deleted in some portion of a GC-rich sequence present in the c-Maf
coding sequence. We presume that this heterogeneity might be the result
of an RT or PCR artifact because of deletions caused by formation of
hairpin loops in this GC-rich sequence.
Of the four remaining PCR bands, two were shown to encode MafB and Nrl.
Although one of the remaining distinct bands was approximately the
expected size for L-Maf, sequence analysis revealed that this band did
not contain L-Maf or indeed any Maf-related product (15 independent
clones were analyzed). Similarly, the sole remaining band also appeared
to be an artifact unrelated to Maf sequence. Thus we were unable to
isolate the mouse homologue of chicken L-Maf from lens RNA in this analysis.
c-Maf Expression Starts in Head Ectoderm Destined to Become the
Lens Vesicle--
To determine the temporal and spatial expression
profiles of the three large Maf factors we detected in the lens, we
performed in situ hybridization analyses on both whole mount
and thin-sectioned e9.0 to e14.5 mouse embryos. Two independent RNA
probes were prepared from the 5' and 3' end regions of the c-Maf
cDNA. From whole-mount in situ analysis, we found that
c-Maf mRNA was expressed in the midline of the forebrain (Fig.
1A) and in the eye region
(Fig. 1B) of e9.0 embryos. In e10.0 to e10.5 embryos, c-Maf
expression in the developing lens progressively intensified, whereas
expression from the midline diminished with age. Embryos were also
hybridized with sense probes, but we did not detect any substantial
signals (data not shown). We performed this whole mount in
situ analysis 4 times, and a total of 35 embryos were hybridized
with the antisense probes, and 10 embryos were hybridized with the
sense probes. The results were quite reproducible.
In the thin section in situ analysis, c-Maf mRNAs were
localized to the head ectoderm destined to become the lens vesicle in
e10.0 embryos (Fig. 2A). It is
of interest to note that c-Maf mRNA was already present in the head
ectoderm of e9.0 embryos, before the overlying ectoderm began to
invaginate, and was also detected in the lens placode. By e10.5, the
lens placode, which now strongly expressed c-Maf, had folded inward to
form the vesicle (Fig. 2B). At e11.5, c-Maf continued to be
abundantly expressed in the lens. The expression was much stronger in
the primary fiber cells (Fig. 2, C (arrow) and
D, shows a sense probe control) than in epithelial cells and
was also prominent in the neural tube (Fig. 2, E
(arrowheads) and F, shows a sense probe
control).
The expression of MafB and Nrl was also examined by in situ
analyses. MafB mRNA was found in lens epithelial cells in e10.5 to
e14.5 embryos but not in the lens fiber cells (Fig. 2, G and H, and data not shown). On the other hand we found that Nrl
mRNA was not expressed during the early stages of the lens
development (Ref. 20 and data not shown). In summary, these data show
that c-Maf and MafB are expressed in the developing lens with distinct distribution profiles, suggesting that they may play important but
distinct roles in lens differentiation.
Cloning and Structural Analysis of c-maf Gene--
To enable the
analysis of c-maf mutant mice, we screened a 129/SVJ mouse
genomic DNA library using a mouse c-Maf cDNA probe. Of 16 clones
recovered, 6 were found to encode the entire ORF of the
c-maf gene. Restriction enzyme site mapping and sequence analysis indicated that the c-maf ORF is uninterrupted by
introns (Fig. 3A).
To determine the transcription start site, we performed 5'-RACE
analysis using e12.5 mouse embryonic RNA. Two cDNA species were
recovered; one category of RACE clones was 144 base pairs longer than
the second and extended the 5' end of the known cDNA sequence
(marked by an asterisk) by 15 base pairs (Fig.
3B). We therefore designated this site as the transcription
initiation site. When compared with the genomic sequence, these RACE
clones showed no evidence for a distinct first exon, again suggesting that the c-maf gene is composed of but a single exon. A MARE
motif was identified at Gene Targeting of the c-maf Locus--
To disrupt c-maf
in ES cells, a targeting construct was prepared to replace virtually
the entire gene ORF with the lacZ and neo genes
(Fig. 3C). ES cells were electroporated with the targeting construct, and neomycin-resistant cells were selected in
G418-containing medium. Of 360 G418-resistant clones screened, 11 clones had undergone homologous recombination at the c-maf
locus. Five independent clones were injected into blastocysts, and male
chimeras were generated that transmitted the c-maf mutation
to their offspring. Genotyping of progeny was performed by PCR and
Southern blot hybridization. Both methods clearly identified homologous
recombinants in the c-maf locus (Fig. 3, D and
E).
Expression of the LacZ Gene in Heterozygous Mutant
Mice--
Staged embryos and adult tissues from c-maf
heterozygous mutant mice were stained for LacZ activity. LacZ
expression was strongly detected in the lens, kidney, and brain (data
not shown). In the developing lens, LacZ expression was first detected
in the lens primordium of head ectoderm at e9.5 and was restricted to
the lens vesicle by e10.5 (Fig. 4,
A and B), which was in good agreement with the
in situ hybridization results.
By e12.5, LacZ expression was confined to the primary lens fiber cell
(Fig. 4C). Signal was also detected in optic nerve (Fig. 4C). LacZ expression was also extensive in the lens fiber
cells of e14.5 embryos (Fig. 4D). Importantly, the reporter
gene was expressed exclusively in lens fiber cells, but not lens
epithelial cells, in the adult (Fig. 4, E and F).
Under higher magnification, LacZ staining in the adult lens was
exclusive to secondary lens fiber cells (Fig. 4F). These
data correlate well with the results from the in situ
analysis (above). The data suggested that c-Maf may play an important
role in lens fiber cell development.
c-maf Homozygous Mutant Mice Lack Normal Lens
Structure--
c-maf heterozygous mutant mice, derived from
two different ES cell clones and maintained in either C57BL/6J × 129/SV or ICR × 129/SV hybrid genetic backgrounds, were intercrossed.
106 embryos, newborns, and one-month-old mice were collected from 17 litters (Table I). Five c-maf
homozygous null mutant newborns were recovered. Although this was lower
than the expected Mendelian ratio, it indicated that c-maf
homozygous mutants could complete gestation. However, no homozygous
mutant mice were found among the 1-month postnatal group, indicating
that the lack of c-maf resulted in complete postnatal
lethality. The etiology of this peri- and postnatal lethality remains
to be elucidated.
Examination of c-maf homozygous mutant newborn mice revealed
that they lacked normal lens structures (Fig.
5A; B is a wild type control). From microscopic histological examination, we failed to
detect normal elongation of the lens fiber cells in the
c-maf (
To better define the time lens malformation in c-maf mutant
animals was first apparent, we examined e11.5 and e16.5 embryos. The
lens structure in e16.5 wild type embryos was visible after formaldehyde fixation, but in c-maf ( Crystallin Gene Expression Is Disrupted in the c-Maf-null Mouse
Lens--
One plausible explanation for the lens malformation in
c-maf knockout mice is that c-Maf is necessary for the
expression of crystallin genes during lens fiber cell development. To
address this question, we examined the expression of crystallin genes using anti-mouse
In the lens of c-maf mutant heterozygous newborns, these
antibodies clearly detected the respective crystallins; the signals were specifically restricted to lens (Fig.
6). In contrast, the anti-crystallin
antibody staining was markedly reduced in the lens of c-Maf-null
newborns. In the c-Maf-null newborns, there was a layer of primary lens
fiber cells despite the absence of a typical lens structure. An
important finding here is that the primary lens fiber cells were
immunoreactive to the anti-
Enucleated anti-crystallin immunoreactive cells were also found in the
lens cavity of c-Maf-deficient newborns. These cells are strongly
immunoreactive with the
To detect crystallin expression more sensitively, we also performed
RT-PCR analysis. Total RNA was extracted from the heads of e16.5
embryos, and mRNA for each class of crystallin was amplified using
specific PCR primer pairs (28). We found that the expression of all
crystallin genes was significantly reduced, and in particular, the
expression of From the RT-PCR, in situ hybridization, and LacZ
staining analyses, we showed here that the c-Maf transcription factor
is expressed specifically in lens fiber cells during embryonic lens development. This expression persists throughout gestation and continues into adulthood. Targeted disruption of c-maf
resulted in a specific block in lens fiber cell differentiation,
consequently leaving the lens cavity empty. These results demonstrate
that c-Maf is required for proper lens development.
In contrast to the c-maf-deficient mice, the Elo
(eye lens obsolescence) mutant mouse suffers from inviability of
central lens fiber cells. The affected and malformed central fiber
cells necrosed and completely collapsed, leaving the lens vesicle
partially open (29). In the Elo mutant mouse, there is a
frameshift mutation in one of the crystallin genes, The expression of Two other large Maf family factors, MafB and Nrl, are also expressed in
the mouse lens (Ref. 20 and this study), therefore prompting the
question as to how Maf family transcription factors individually or
cooperatively execute their roles during vertebrate lens formation. An
important finding here is that MafB mRNA is expressed exclusively
in lens epithelial cells, whereas Nrl is expressed widely in the lens
but at a much later stage of lens development than c-Maf and MafB.
Compared with the other two large Maf proteins, c-Maf is unique in that
its expression is restricted to lens fiber cells after the formation of
the lens structure. The complementary distribution of c-Maf and MafB in
the lens fiber and epithelial cells, respectively, is intriguing and
suggests that each large Maf factor has a unique role in vertebrate
lens development.
A natural MafB mouse mutant, kreisler (kr), has previously
been described that lacks normal rhombomere formation and inner ear
structure (31). However, no phenotype has been reported in the eyes or
lens of the kr mouse. It was shown recently that the
kr mutant mouse is not a MafB-null mutant, as MafB activity persists in many functional aspects (32). Therefore, the role of MafB
in the differentiation and function of lens epithelial cells remains to
be clarified.
Another large Maf factor, L-Maf, was isolated from the chicken and was
shown to be a key regulator of We would like to thank Drs. K. Eto, F. Grün, H. Hoshino, S. Ishibashi, M. Kajihara, F. Katsuoka, K-C.
Lim, T. Murata, T. H. Momose, H. Motohashi, N. Osumi, N. Kaneko,
K. Umesono, K. Yoh, and R. T. Yu for their help. We also thank Dr.
M. Taketo for providing pPGK DT-A plasmid.
*
This work was supported in part by grants-in-aid from the
Ministry of Education, Science, Sports, and Culture, Core Research for
Evolutional Sciences and Technology, and the Japanese Society for
Promotion of Sciences.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
This paper is dedicated to the memory of Dr. Kazuhiko Umesono, whose
life work was dedicated to elucidating the multiple facets of
transcription factor research.
**
To whom correspondence should be addressed: Center for TARA,
University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan. Tel.:
81-298-53-6158; Fax: 81-298-53-7318; E-mail: masi{at}tara.tsukuba.ac.jp.
2
H. Ogino and K. Yasuda, unpublished observation.
3
K. Yasuda, unpublished observation.
The abbreviations used are:
MARE, Maf responsive
element;
ES, embryonic stem;
ORF, open reading frame;
LacZ, Escherichia coli
Regulation of Lens Fiber Cell Differentiation by Transcription
Factor c-Maf*
§¶,,,,
,**
Center for Tsukuba Advanced Research
Alliance and Institute of Basic Medical Sciences, Scripps Research Institute,
La Jolla, California 92037
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (lacZ) gene into the c-maf locus allowed
visualization of c-Maf accumulation in heterozygous mutant mice by
staining for LacZ activity. Homozygous mutant embryos and newborns
lacked normal lenses. Histological examination of these mice revealed
defective differentiation of lens fiber cells. The expression of
crystallin genes was severely impaired in the c-maf-null
mutant mouse lens. These results demonstrate that c-Maf is an
indispensable regulator of lens differentiation during murine development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A-crystallin gene has been identified (11,
12). Biochemical analyses of the core region of this enhancer revealed
key interacting transcription factors (13, 14). Of the cis
elements identified in the enhancer, the CE2 sequence, which shares
high similarity with the Maf responsive element (MARE1
(15)), is crucial for its transcriptional activity. MARE-related consensus sequences have also been found in the regulatory regions of
other lens-specific genes (12).
CE2 enhancer element, was isolated from chicken lens (13). L-Maf is
a member of the large Maf oncoprotein/transcription factor family
(16-18). The Maf family factors contain a basic leucine zipper domain
and bind to MARE either as homodimers or as heterodimers with other
basic leucine zipper transcription factors (19). L-Maf regulates the
expression of multiple lens-specific genes, and its forced expression
can convert primary chick embryonic neural retina cells in culture to a
lens fiber cell fate, indicating that vertebrate lens induction and
differentiation can be triggered by the ectopic expression of L-Maf
(13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 to 402 and 753 to
1127) of the c-maf gene were subcloned, and sense and
antisense probes were synthesized using the T3 and T7 RNA polymerases
with (digoxigenin)-UTP labeling kit (Roche Molecular Biochemicals).
5 to 402 of c-Maf was
used as probe.
433/507 and
700/725 with respect to the mRNA initiation site, respectively. Primer 2 was used in nested PCR. The resultant PCR fragments were subcloned into TA vector (Promega) and sequenced.
-D-galactopyranoside (X-gal).
A-, B-, -, and 
-crystallin monoclonal antibodies (27).
Sections were then incubated with biotin-conjugated goat anti-mouse IgG
and avidin-alkaline phosphatase.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of c-maf expression
by whole mount in situ hybridization. c-Maf
mRNA accumulation was analyzed by in situ hybridization
to e9.0 to e11.5 murine embryos. A, sagittal views of
embryos hybridized with antisense c-Maf probes. Note that c-Maf
mRNA is expressed in the lens and along the midline of the early
forebrain (arrow in e9.0). B shows a higher
magnification of A.
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Fig. 2.
Thin tissue section in situ
hybridization analysis of c-Maf and MafB expression. c-Maf
mRNA is detected in e10.0 lens placode (indicated by the
arrow in A) and e10.5 lens vesicle
(B). At e11.5, c-Maf expression is detected in the
elongating lens fiber cells (C (arrow), and
D shows a sense probe control) and on the ventral side of
the neural tube (E, arrowhead) and F,
sense probe). MafB mRNA is restricted to lens epithelial cells at
e14.5 (G, arrow). H shows a higher
magnification of G. Co, cornea; Fi,
lens fiber cells; LV, lens vesicle; OC, optic
cup; Re, retina.
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Fig. 3.
Structure and targeted disruption of
c-maf gene. A, restriction enzyme map
of c-maf gene. B, sequence of the
c-maf proximal promoter and upstream region. The 5' end of
the reported c-Maf cDNA sequence is indicated by an
asterisk. The transcription start site is shown as +1, and
the rest of the sequence is numbered from this site. C,
schematic representations of the wild type allele, targeting vector,
and mutant allele are shown. The solid box in the wild type
allele represents the coding sequence. Restriction enzyme sites are
E, EcoRI; S, SalI;
M, MscI; H, HindIII;
N, NcoI; X, XhoI. The
positions of wild type and mutant allele-specific 5' primers and the
common 3' primer used in the genotyping analysis are indicated.
D, genotyping of four mutant ES cell clones heterozygous for
c-maf gene targeting by both Southern blotting and PCR. The
selected clones are 44 (lane 1), 144 (lane 2),
153 (lane 3), and 194 (lane 4). E, an F2 litter
was genotyped by both Southern hybridization and PCR. This litter had 2 wild types (lanes 4 and 7), 1 homozygous mutant
(lane 1), and 4 heterozygous mutants (lanes 2,
3, 5, and 6) neonatal pups.
WT, wild type allele; kbp, kilobase pairs;
MT, mutant allele.
47 to 38, immediately 3' to the putative
TATA box, in the proximal promoter region. We also performed Southern blot analysis on high molecular weight DNA, and restriction enzyme mapping showed only the expected genomic fragment sizes, suggesting that the uninterrupted locus we had cloned was not a pseudogene and
that c-maf exists as a single copy gene in the mouse genome (data not shown).
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Fig. 4.
LacZ reporter gene expression during lens
development of heterozygous c-maf mutant mice.
LacZ expression (arrowhead) is detected at e9.5 in the head
ectoderm region where the optic cup (arrow) contacts
(A). LacZ activity is also detected in the lens fiber cells
of e10.5 to e14.5 c-maf heterozygous mutant embryos
(B-D) and in the lens of 9-week-old adult mouse
(E). Higher magnification of the adult lens shows that the
epithelial cell layer exhibits no LacZ staining (F,
arrow). Fi, lens fiber cells; LV, lens
vesicle; Re, retina.
Progeny recovered from heterozygous c-maf mutant intercrosses
/) lens (Fig. 5C), which were properly
formed in wild type littermates (Fig. 5D).
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Fig. 5.
c-maf-deficient mice lack normal lens
structure. Homozygous c-maf mutant newborns lack a
normal lens structure (A, arrow; B
shows wild type). c-Maf deficiency prevents elongation of posterior
lens fiber cells in c-maf homozygous mutant (C)
but not in wild type (D) animals. A normal lens structure is
not visible in the e16.5 c-maf homozygous mutant embryo
(E, arrow), whereas the wild type e16.5 embryo
has a visible lens structure (F). In the e16.5
c-maf null mutant embryo (G), unlike its wild
type littermate (H), the lens fiber cells are not elongated,
and consequently, the lens cavity is empty. Sections were stained with
hematoxylin and eosin. Original magnification of the sections are ×200
(C, G, and H) or ×100
(D).
/) embryos, no
normal lens structure was evident (Fig. 5E; F
shows a wild type littermate), and there was no elongation of lens
fiber cells (Fig. 5G, H shows wild type).
However, in e11.5 embryos, the lens vesicle was beginning to form,
indicating that lens development seems to progress normally despite the
absence of the c-Maf protein (data not shown). In other eye structures,
the retinal layer or pigment epithelium may be affected to some extent,
but further analysis will be necessary to determine whether this is
caused directly by c-Maf deficiency or as a secondary consequence of
impaired lens formation (Fig. 5G).
A-, B-, -, and -crystallin monoclonal
antibodies (27).
B- and -crystallin antibodies (Fig. 6,
B and C, arrows). Also, when higher concentration
of anti-A-crystallin antibody was employed, positive signals were
detected (data not shown). Thus, although the - and -crystallin
genes are under the positive regulation of c-Maf, they can be weakly
transcribed without c-Maf. However, the expression of -crystallin,
known to be specifically expressed in lens fiber cells, was not
detected in the c-Maf-null mouse lens, even at the highest
concentrations of antibody used (Fig. 6D). Thus
-crystallin gene transcription is under strict c-Maf regulatory
control.
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Fig. 6.
Expression of crystallins in the lens of
c-maf heterozygous and homozygous mutant mice.
Immunohistochemical analysis was performed using monoclonal antibodies
that recognize specifically mouse A-, B-, -, and
-crystallins (A to D). E shows a
negative control using bovine serum albumin instead of a primary
antibody. Antibodies against A-, B-, and -crystallins were
used at 100-fold dilution, whereas neat anti--crystallin antibody
was used. The arrows in B and C
indicate signals in primary lens fiber cells. Note that in the
c-maf (/)lens, degenerating cells are present
(B, arrowhead). Fi, lens fiber cells;
E, lens epithelial cells.
B-crystallin antibody (arrowheads in Fig. 6B) and may represent primary lens fiber cells whose
maturation was arrested by the c-Maf deficiency.
-crystallin mRNA apparently disappeared (Fig. 7). In summary, these results thus
demonstrate that c-Maf positively regulates the expression of
crystallin genes during lens fiber cell differentiation.
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Fig. 7.
RT-PCR analysis of crystallin gene expression
in the lens of c-maf heterozygous and homozygous
mutant mice. mRNA for each class of crystallin was amplified
using specific PCR primer pairs (28), and total RNA was extracted from
the heads of e16.5 embryos. A set of primers that amplifies
hypoxanthine phosphoribosyltransferase (HPRT (28)) was used
as an internal control. To identify E and F, the PCR products
amplified with same set of primers were digested with BglII
(E, 149 and 103 base pairs; F, 252 base pairs).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
E-crystallin
(30). Interestingly, a MARE motif that is similar to the CE2 element
has also been found in the lens-specific regulatory region of each
-crystallin gene (12, 28), suggesting that -crystallin genes are
the target genes of c-Maf. However, lens fiber cell differentiation in
the c-maf-deficient mouse is arrested before central fiber cells are formed, thus precluding the possible observation of an
Elo phenotype in the c-maf mutants.
-crystallins could not be detected by
immunohistochemistry or by RT-PCR in c-Maf-deficient newborns or e16.5
mutant embryos, respectively, further supporting the notion that c-Maf
is an important transcriptional regulator of the -crystallin genes.
In addition, the expression of A-, B-, and -crystallins were
found to be down-regulated in the c-Maf-deficient mice. Similar observations were reported recently in the targeted disruption sox-1 mutation (28). Although Sox-1 is indispensable for
mouse lens fiber cell differentiation, crystallins are expressed in the
sox-1 (/) lens. Careful comparison of the
c-maf- and sox-1-deficient mouse lenses revealed
that the c-maf deficit results in a more profound defect in
crystallin gene expression than does the sox-1 deficiency.
In addition, A-and B-crystallin gene-targeted mutations do not
lead to a severe defect in lens formation (6). These observations raise
the question as to whether the decrease or absence of crystallin gene
expression is the main cause of lens malformation in the
c-Maf-deficient mouse. From the analysis of crystallin gene expression
presented here, it is probable that other lens-specific genes that are
also under the regulatory influence of c-Maf and the consequent lack of
this gene product(s) may be responsible for the differentiation block
in lens fiber cells in c-Maf-deficient mice.
A-crystallin gene transcription in
the lens (13). Because MARE motifs are found in the regulatory regions
of many lens-specific genes, Maf family transcription factor are
probably required for multiple aspects of normal lens development. In
the chicken, L-Maf appears to be the prime target of a lens induction
signal emanating from the optic vesicle (13). Although we did not
detect a murine homologue of L-Maf in our experiments, the existence of
four distinct large Maf factors has already been demonstrated in
Xenopus.3
Therefore the existence of a murine L-Maf gene is plausible. The
differential functional contributions of c-Maf, L-Maf, and other large
Maf transcription factors to vertebrate lens formation are yet to be
fully elucidated.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-galactosidase gene;
5'-RACE, 5'-rapid
amplification of the cDNA end;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
DT-A, diphtheria toxin
A.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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