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Molecular and Cellular Biology, April 2000, p. 2498-2504, Vol. 20, No. 7
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Expression and Functional Analysis of
Uch-L3 during Mouse Development
Laurie Jo
Kurihara,
Ekaterina
Semenova,
John M.
Levorse, and
Shirley M.
Tilghman*
Howard Hughes Medical Institute and
Department of Molecular Biology, Princeton University, Princeton,
New Jersey 08544
Received 16 December 1999/Accepted 21 December 1999
 |
ABSTRACT |
Mice homozygous for the s1Acrg deletion at
the Ednrb locus arrest at embryonic day 8.5. To determine
the molecular basis of this defect, we initiated positional cloning of
the s1Acrg minimal region. The mouse
Uch-L3 (ubiquitin C-terminal hydrolase L3) gene was mapped
within the s1Acrg minimal region. Because
Uch-L3 transcripts were present in embryonic structures
relevant to the s1Acrg phenotype, we created a
targeted mutation in Uch-L3 to address its role during
development and its possible contribution to the s1Acrg phenotype. Mice homozygous for the
mutation Uch-L3
3-7 were viable, with no
obvious developmental or histological abnormalities. Although high
levels of Uch-L3 RNA were detected in testes and thymus,
Uch-L33-7 homozygotes were fertile, and no
defect in intrathymic T-cell differentiation was detected. We conclude
that the s1Acrg phenotype is either complex and
multigenic or due to the loss of another gene within the region. We
propose that Uch-L3 may be functionally redundant with its
homologue Uch-L1.
| |
INTRODUCTION |
The analysis of induced mutations
has proven to be a powerful method for identifying genes involved in
development in many species. The first genetic screen for induced
mutations in the mouse was the specific locus test (SLT)
(22; for a review, see reference
19). With a variety of mutagens, the SLT generated new alleles over seven tester loci chosen for their easily scored mutant phenotypes. The molecular lesions ranged in size from single base changes to large deletions spanning multiple centimorgans. These
alleles have been useful in the positional cloning of the genes
underlying the tester loci themselves, such as short
ear/Bmp5 (11). In addition, large deletion alleles have
also been used to assign biological function to chromosomal regions
flanking the specific loci. For the albino-linked deletion
region required for embryonic ectoderm development (eed),
the corresponding gene was identified through positional cloning
(27).
The piebald (s) locus was one of the specific
loci used in the SLT. piebald encodes the endothelin B
receptor (EDNRB), a G protein-coupled seven-transmembrane receptor
required for the migration of two neural crest derivatives, melanocytes
and enteric ganglia (8, 26). Mice homozygous for a null
allele of Ednrb are amelanocytic and develop megacolon,
resulting in juvenile lethality (12, 15). Many
Ednrb alleles generated in the SLT are deletions that
exhibit a more severe phenotype than the loss of Ednrb
alone, most likely due to the loss of linked essential genes
(16). Through phenotypic analysis of individual deletions combined with molecular mapping of deletion breakpoints and
complementation analysis of deletion alleles, chromosomal regions
associated with distinct developmental defects were defined
(17). These include embryonic lethality, neonatal lethality,
and skeletal and central nervous system defects.
The s1Acrg deletion results in embryonic
lethality; based on complementation analysis, the portion of the
deletion associated with this defect was defined as the
s1Acrg minimal region (17). Embryos
homozygous for s1Acrg arrest at embryonic day
8.5 and display a complex phenotype that includes cranial neural tube
defects, altered somite and notochord morphology, and a failure to
complete embryonic turning and heart looping morphogenesis (T. P. O'Brien, personal communication). Based on histological and molecular
marker analyses, this phenotype results from defects that are already
evident in the primitive streak and node. Although the
s1Acrg deletion phenotype may be multigenic,
several single-gene mutations lead to arrest at embryonic day 8.5 with
a similar phenotype (for a review, see reference 3).
Therefore, the s1Acrg phenotype could result
from the loss of a single gene that is essential during development.
To determine the molecular basis of the s1Acrg
phenotype, we initiated an analysis of the genes within the minimal
region. For this purpose, a 1.4-Mb contig of P1, bacterial artificial
chromosome (BAC), and yeast artificial chromosome clones was
constructed (L. J. Kurihara, E. Semenova, D. L. Metallinos,
X.-J. Guan, R. S. Ingram, A. Goddard, and S. M. Tilghman,
unpublished data). Based on the low CpG content of syntenic human
chromosome 13 (6) and the small (compared to other
chromosomes) number of human expressed sequence tags (ESTs) mapping to
chromosome 13 (24), we predicted that the
s1Acrg region is gene poor. Indeed, no CpG
islands were identified within the contig. However three ESTs were
mapped by sequence analysis of a single CpG-rich BAC clone. In
addition, two human genes that map proximal to EDNRB
cross-hybridized to the mouse s1Acrg contig
(Kurihara et al., unpublished data). One of these genes is human
UCH-L3, which encodes ubiquitin C-terminal hydrolase L3.
The ubiquitin pathway is constitutive and essential for the turnover of
many short-lived regulatory proteins as well as damaged proteins (for a
review, see reference 18). However, mutations within
ubiquitin pathway enzymes have revealed distinct phenotypes due to
either their substrate specificity or particular spatial or temporal
expression patterns. Moreover, certain mutations have indicated a role
for the ubiquitin pathway during development. For example, loss of the
mouse UbcM4 ubiquitin-conjugating enzyme leads to embryonic
lethality (7), the Caenorhabditis elegans let-70/ubc-2 ubiquitin-conjugating enzyme is essential for larval development (32), and mutation of the Drosophila fat
facets deubiquitinating enzyme leads to defects in eye cell fate
determination (9).
Here we report the characterization of the mouse Uch-L3 gene
and show that its expression pattern during embryogenesis makes it a
candidate for a gene underlying the s1Acrg
defect. To directly test whether the absence of Uch-L3 alone leads to embryonic lethality, we generated a targeted mutation in this gene.
| |
MATERIALS AND METHODS |
Isolation of Uch-L3.
A human EST corresponding to a
UCH-L3 cDNA was shown by low-stringency hybridization to map
to a BAC contig of the s1Acrg minimal region.
The human UCH-L3 probe was used to isolate mouse cDNAs from
an embryonic day 17.5 
gt11 library (Clontech). Phage inserts from
purified clones were amplified by PCR, cloned into the TA vector
(Invitrogen), and sequenced with an ABI Prism labeling kit using an ABI
373 sequencer. Two partial cDNAs (mUCH4 and mUCH12) and one full-length
cDNA (mUCH14) were isolated.
Expression analysis.
Whole-mount in situ hybridization to
embryos was performed as described by Wilkinson and Nieto
(30). Digoxigenin-labeled RNA probes were synthesized with
T7 polymerase. The antisense Uch-L3 probe included exons 3 to 10 from mUCH14 linearized with StuI. The sense control
probe included exons 1 to 8 from mUCH4 linearized with
BglII.
Total RNA was extracted from mouse tissues with Trizol (GIBCO/BRL).
Fifteen micrograms of RNA was separated in 1% agarose gels containing
morpholinepropanesulfonic acid (MOPS)-formaldehyde and transferred to
Hybond N+ membranes (Amersham). Blots were hybridized in Church buffer
(2) at 65°C and washed in 0.1× SSC (1× SSC is 0.15 M
NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate at 23 and 65°C. Radiolabeled probes were synthesized from fragments of
Uch-L3 (wild-type mUCH4 and mutant 
3-7
[Uch- L33-7]) and 
-actin cDNA clones.
Reverse transcription (RT)-PCR was performed with a cDNA cycle kit
(Invitrogen). Primers used to amplify
Uch-L33-7 RNA were
5'-ATGGAGGGTCAACGCTGGCT-3' and
5'-GGTGTTTCTGTCAAGATGCTAT-3'.
PCR products were cloned with
a TOPO-TA kit (Invitrogen) and sequenced
with the ABI Prism labeling
kit using an ABI 373
sequencer.
Generation and analysis of Uch-L33-7
mutant mice.
To delete the 9.5-kb region encoding exons 3 to 7, two flanking genomic DNA fragments were subcloned into the targeting
vector pLOX-PNT, which contains the neomycin resistance gene driven by the phosphoglycerol kinase 2 promoter (PGK-NEO) and herpes simplex virus thymidine kinase (25). Targeting arms were subcloned
from a BAC containing Uch-L3 into the Bluescript vector, where
polylinker restriction sites and HindIII/KpnI
adapters were utilized for subsequent cloning into pLOX-PNT. The
targeting arms included a 3.25-kb SpeI fragment upstream of
exon 3 at the 5' end and a 4-kb HindIII fragment
downstream of exon 7 at the 3' end.
The
Uch-L33-7 targeting construct was
linearized at a unique
NotI site and electroporated into CJ7
embryonic stem (ES) cells
(
28), followed by selection with
125 µg of active G418 (Sigma)
per ml and 1 µM ganciclovir (Roche).
Following colony purification,
ES cell DNA was extracted and digested
with either
HindIII (5'
arm) or
PstI (3'
arm), separated in 1% agarose-Tris-borate-EDTA
(TBE) gels, and
transferred to Hybond N+ membranes. Radiolabeled
probes were PCR
products generated from genomic DNA flanking each
targeting arm,
denoted 5' and 3' probes. Correctly targeted ES
cell clones were
obtained at a frequency of one in nine G418-selected
colonies.
Three independent ES cell clones (A2, F3, and C11) were injected into
C57BL/6 blastocysts and implanted into pseudopregnant
mice. Chimeras
were bred to C57BL/6 mice, and their agouti progeny
were genotyped. PCR
genotyping was performed on tail DNA with
a common forward primer from
the genomic sequence flanking the
deletion
(5'-GGAACTACTGAGCCATATGTGC-3'). This primer was used
with
either a reverse primer derived from endogenous DNA within
the deletion
for detecting the wild-type allele (5'-CCGACTTACTCCATCACTTCAC-3')
or a reverse PGK primer from the NEO cassette for detecting the
targeted allele (5'-CTTGTGTAGCGCCAAGTGC-3'). PCR conditions
were
35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min.
Fluorescence-activated cell sorter (FACS) analysis was performed
on thymus and spleen cells as described by Beavis and Pennline
(
1).
| |
RESULTS AND DISCUSSION |
Isolation of mouse Uch-L3.
Ednrb maps at 51 cM on
mouse chromosome 14, a region that is syntenic with human chromosome
13q22. As the s1Acrg region maps immediately
proximal to Ednrb, we searched the National Center for
Biotechnology Information human gene map for genes linked to
human EDNRB and identified UCH-L3. We
then found that the human UCH-L3 cDNA cross-hybridized to
the BAC contig over the s1Acrg minimal region
(Kurihara et al., unpublished data). To isolate the mouse
Uch-L3 gene, the human UCH-L3 probe was used to
screen a mouse cDNA library. Sequence analysis of mouse
Uch-L3 cDNAs revealed an ~900-nucleotide transcript with a
predicted open reading frame encoding 230 amino acids (Fig.
1). The predicted mouse UCH-L3 protein
displays 96% identity to its human orthologue Uch-L3 and 52% identity
to its mouse paralogue UCH-L1 (Fig. 2).
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FIG. 1.
Uch-L3 gene sequence and structure. Exon (Ex)
boundaries are denoted above the nucleotide sequence. The exon 4-exon 5 boundary was ambiguous, as indicated. The start and stop codons and
residues deleted in Uch-L33-7 are underlined;
the caret denotes conserved cysteine 95.
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FIG. 2.
Alignment of the mouse (m.) UCH-L3 amino acid sequence
with human (h.) Uch-L3 and mouse UCH-L1. Identical residues are boxed
and darkly shaded, and conserved changes are boxed and lightly shaded.
Dashes indicate gaps relative to mUCH-L3.
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|
The UCH family of deubiquitinating enzymes consists of two members,
UCH-L1 and UCH-L3. This small conserved family differs
from the larger
and highly diverse UBP family of deubiquitinating
enzymes (for
reviews, see references
4 and
31). Although
enzymatic activity has been confirmed
for members of both families
in vitro, the in vivo substrate
specificity and function of the
majority of these enzymes remain
unknown. Recently, a mutation
in
UCH-L1 was linked to
Parkinson's disease in humans (
14) and
to the gracile
axonal dystrophy (
gad) mutation in mice (
23).
Because the loss of
Uch-L1 results in the accumulation of
protein
aggregates, leading to neurodegeneration, the likely in vivo
function
of
Uch-L1 is to stimulate protein degradation in
neurons where
it is primarily
expressed.
Expression of Uch-L3.
To consider Uch-L3 as a
candidate gene for s1Acrg-dependent embryonic
lethality, the expression of Uch-L3 at embryonic day 8.5 was
verified by RT-PCR (data not shown). In addition, Uch-L3
transcripts were found within structures relevant to the
s1Acrg phenotype by whole-mount in situ
hybridization (Fig. 3). These include the
edges of the open neural folds, which fail to close in
s1Acrg mice, and the somites, which are
disorganized. Uch-L3 transcripts were also present in
structures that form after embryonic day 8.5, including the rim
of the posterior neuropore, the apical ectodermal ridge of the limb
buds, the branchial arches, the somites, and the tail bud. Combined
with its location in s1Acrg, this expression
pattern is consistent with a role for Uch-L3 during
embryogenesis.
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FIG. 3.
Analysis of Uch-L3 transcripts by
in situ hybridization. (A) An embryonic day 8.5 (e8.5) embryo is
stained at the open edge of the anterior and posterior neural folds
(arrow). Staining throughout the embryo was also detected. (B) An e9.5
embryo shows staining at the rim of the posterior neuropore (arrow).
(C) An e10.5 embryo is stained at the branchial arches (arrowhead),
apical ectodermal ridge (arrow), somites, and tail bud. (D) An e10.5
embryo hybridized with the control sense Uch-L3 probe shows
no staining.
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To characterize the expression of
Uch-L3 in adult mice,
Northern analysis of RNAs isolated from multiple organs was performed
(Fig.
4). The
Uch-L3
transcript was ~900 nucleotides long, as
predicted by the cDNA
sequence. Although
Uch-L3 RNA was detected
in all tissues
analyzed, particularly high levels were present
in the testes and to a
lesser degree in the thymus. This result
suggests that
Uch-L3 may also have a role in adult mice, particularly
during spermatogenesis or intrathymic T-cell differentiation,
both of
which are dependent on the ubiquitin pathway.
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FIG. 4.
Uch-L3 expression in adult tissues.
Total RNAs from the tissues indicated were hybridized to both
Uch-L3 and -actin probes. The order of the lanes, from
left to right, is thymus, gut, lung, liver, spleen, kidney, testis,
heart, tongue, brain, and placenta. Based on ethidium bromide
staining of rRNA bands, relatively equivalent amounts of RNA were
loaded in each lane (data not shown).
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Generation and analysis of Uch-L33-7
mice.
To directly address the role of Uch-L3 during
development, mice with a targeted mutation were generated. To design
this allele, we first determined the genomic structure of
Uch-L3 by alignment of the cDNA sequence with corresponding
fragments of the BAC genomic sequence (Fig. 1 and
5a). Restriction mapping of BAC clones
was also used to estimate the size of the Uch-L3 locus at 47 to 60 kb. Exons 1 and 2 are ~100 bp apart; up to 15 kb downstream lie exons 3 to 7, which are clustered within 9.5 kb; and exons 8 to 10 lie
at least 15 kb further downstream.
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FIG. 5.
Construction of a targeted
Uch-L33-7 allele. (a) The genomic structure
of Uch-L3 is indicated in the top line. The wild-type allele
depicts the SpeI (gray box) and HindIII
(hatched box) fragments used as targeting arms flanking exons 3 to 7. The wild-type allele was detected as a 7.5-kb HindIII
fragment with the 5' probe and as an 11-kb PstI fragment
with the 3' probe. The arrows indicate the primers used to detect the
wild-type allele by PCR. The Uch-L33-7 allele
depicts the replacement of exons 3 to 7 with PGK-NEO following
targeting. The targeted allele was detected as a 5.5-kb
HindIII fragment with the 5' probe and as an 8-kb
PstI fragment with the 3' probe. The arrows indicate the
primers used to detect the targeted allele by PCR. (b) Southern blot
hybridization of the 5' and 3' probes to wild-type (+/+) and
heterozygous (+/) mouse DNAs digested with HindIII
(5') or PstI (3') to detect the wild-type and targeted
restriction fragments. (c) Total RNAs from wild-type (+/+) and
Uch-L33-7 (/) testes were hybridized to
Uch-L3 and -actin probes.
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Since only a portion of
Uch-L3 could be targeted due to its
large size, it was most critical to remove residue 95, the catalytic
cysteine that is essential for hydrolase activity in vitro
(
13).
Because the crystal structure of human Uch-L3 predicts
a small,
single-domain hydrolase (
10), it is unlikely that
Uch-L3 possesses
any other enzymatic activity. Therefore, we created a
deletion
of clustered exons 3 to 7 which removed up to 90% of the
protein,
including C95 (Fig.
5a). If exon 2 spliced over PGK-NEO in
frame
to exon 8, the resulting 90-amino-acid protein would still lack
C95 and hydrolase
activity.
Because the
s1Acrg phenotype is recessive,
Uch-L33-7 heterozygotes were bred to
homozygosity. Mice homozygous for
Uch-L33-7
were obtained at weaning at the expected Mendelian frequency.
To assess
the transcripts from
Uch-L33-7, Northern
analysis and RT-PCR were performed. As shown in Fig.
5c, a truncated
transcript was present in homozygotes at a level
equivalent to that of
the wild-type transcript. RT-PCR revealed
that the
Uch-L33-7 RNA was composed of two products.
One, which included exons 1
and 2 spliced in frame to exons 8 to 10, would be capable of encoding
a 90-amino-acid fusion protein. The other,
which included exons
1 and 2 spliced out of frame to exons 9 and 10, would encode only
the first 18 amino acids of the protein. These
results confirm
that mice lacking functional
Uch-L3 are
viable. Furthermore, we
generated
Uch-L33-7/s1Acrg compound
heterozygotes to determine whether the loss of
Uch-L3,
together with a haploid copy of
s1Acrg, would be
deleterious. However, the offspring were viable and
fertile. While we
cannot rule out the possibility that the loss
of
Uch-L3
contributes to the
s1Acrg phenotype, its loss
alone cannot account for embryonic lethality.
Thus, the
s1Acrg phenotype is either complex and
multigenic or due to another
gene within the minimal
region.
Mice homozygous for
Uch-L33-7 developed to
maturity with no obvious abnormalities. Although
Uch-L3 is
expressed in embryonic
structures required for skeletal patterning, no
abnormalities
were identified in specimens of
Uch-L33-7 neonates that were stained with
alcian blue-alizarin red and
cleared to view cartilage and bone (data
not shown). Particular
attention was paid to the axial skeleton, limbs,
and craniofacial
structures, which are derived from
Uch-L3-expressing embryonic
tissues. Similarly, although
Uch-L3 is expressed in many adult
tissues, no histological
defects were observed in hematoxylin-eosin-stained
sections of mutant
kidney, spleen, thymus, lymph node, intestine,
liver, lung, adrenal
gland, testis, ovary, brain, or heart (data
not shown). Because high
levels of
Uch-L3 RNA were detected in
wild-type testes and
to a lesser degree in thymus, we determined
whether the functions of
these organs were affected in
Uch-L33-7 homozygotes.
Within the testes, the ubiquitin pathway is required during
spermatogenesis, as shown by the male sterility that is associated
with
the loss of the mouse
HR6B ubiquitin-conjugating enzyme
(
21).
Based on the mutant phenotype,
HR6B appears
to be required during
postmeiotic chromatin condensation, when histones
are replaced
by transition proteins and protamines. However, fertility
and
sperm morphology were unaffected in
Uch-L33-7 homozygous mice (data not
shown).
Within the thymus, differentiation of CD4
CD8
+ T lymphocytes is dependent on the generation of major
histocompatibility complex
(MHC) I peptide antigens by the ubiquitin
pathway (for a review,
see reference
20). For
example, a mutation of the ubiquitin
proteasome component LMP2 leads to
a 49% reduction in CD4
CD8
+ T lymphocytes
within the thymus (
29). A mutation of the proteasome
component LMP7 leads to a 25 to 45% decrease in MHC I cell surface
staining (
5), another event that is dependent on MHC I
peptide
antigens. However, in
Uch-L33-7 mice,
no significant reduction in CD4
CD8
+ T
lymphocytes within the thymus or spleen was observed by FACS
analysis
with CD4, CD8, and T-cell receptor
antisera (Table
1). In addition, MHC I cell surface
staining was not significantly
reduced in
Uch-L33-7 mice when assayed by FACS analysis
with
H-2Kb antiserum (Table
1).
The absence of either an embryonic or an adult phenotype in
Uch-L33-7 mice implies either that
Uch-L3 performs an undetected nonessential
function or that
Uch-L3 is functionally redundant with
Uch-L1. Uch-L3 and
Uch-L1 display 52% identity, and their
expression patterns
overlap in several tissues, including the brain and
testes, where
Uch-L3 is present at high levels. Loss of
Uch-L1 leads to distinct
neurological defects, but it is
possible that the simultaneous
loss of both
Uch-L1 and
Uch-L3 would exacerbate these defects
and result in
additional defects in other organs. Where overlapping
expression
patterns have not been demonstrated, such as during
embryogenesis,
Uch-L3 and
Uch-L1 function would be either
dispensable
or possibly redundant with that of members of the UBP
family of
deubiquitinating enzymes, which do not share sequence
conservation
with the UCH family. However, given the degree of sequence
divergence
between UCH and UBP deubiquitinating enzymes, it is expected
that
they possess distinct substrate specificities. Experiments are
under way to test these
premises.
| |
ACKNOWLEDGMENTS |
We are grateful to Robert S. Ingram for sequencing of cDNA clones
and to Audrey Goddard at Genentech, Inc., for genomic DNA sequencing.
We also thank Se-Ho Park, Albert Bendelac, and Andrew Beavis for
antisera and FACS analysis. The histopathologic analysis of mutant mice
was performed at the University of California Davis Histo-Pathology Laboratory.
L.J.K. was supported by an NRSA award from the National Institutes of
Health. S.M.T. is an investigator of the Howard Hughes Medical Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology, Princeton University, Princeton, NJ 08544. Phone: (609) 258-2900. Fax: (609) 258-3345. E-mail:
stilghman{at}molbio.princeton.edu.
| |
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Molecular and Cellular Biology, April 2000, p. 2498-2504, Vol. 20, No. 7
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
