MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Apr. 2000, p. 2498–2504Vol. 20, No. 7
Expression and Functional Analysis of Uch-L3 during
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
Mice homozygous for the s1Acrgdeletion at the Ednrb locus arrest at embryonic day 8.5. To determine the
molecular basis of this defect, we initiated positional cloning of the s1Acrgminimal region. The mouse Uch-L3
(ubiquitin C-terminal hydrolase L3) gene was mapped within the s1Acrgminimal region. Because Uch-L3
transcripts were present in embryonic structures relevant to the s1Acrgphenotype, we created a targeted
mutation in Uch-L3 to address its role during development and its possible contribution to the s1Acrgphenotype.
Mice homozygous for the mutation Uch-L3?3-7were viable, with no obvious developmental or histological
abnormalities. Although high levels of Uch-L3 RNA were detected in testes and thymus, Uch-L3?3-7homozy-
gotes 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.
The analysis of induced mutations has proven to be a pow-
erful 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 gener-
ated 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 mul-
tiple centimorgans. These alleles have been useful in the po-
sitional cloning of the genes underlying the tester loci them-
selves, 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 devel-
opment (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 break-
points and complementation analysis of deletion alleles, chro-
mosomal regions associated with distinct developmental de-
fects were defined (17). These include embryonic lethality,
neonatal lethality, and skeletal and central nervous system
The s1Acrgdeletion results in embryonic lethality; based on
complementation analysis, the portion of the deletion associ-
ated with this defect was defined as the s1Acrgminimal region
(17). Embryos homozygous for s1Acrgarrest at embryonic day
8.5 and display a complex phenotype that includes cranial
neural tube defects, altered somite and notochord morphol-
ogy, and a failure to complete embryonic turning and heart
looping morphogenesis (T. P. O’Brien, personal communica-
tion). Based on histological and molecular marker analyses,
this phenotype results from defects that are already evident in
the primitive streak and node. Although the s1Acrgdeletion
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 s1Acrgphenotype
could result from the loss of a single gene that is essential
To determine the molecular basis of the s1Acrgphenotype, 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. Metalli-
nos, X.-J. Guan, R. S. Ingram, A. Goddard, and S. M. Tilgh-
man, 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 s1Acrgregion 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 s1Acrgcontig (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 path-
way during development. For example, loss of the mouse
UbcM4 ubiquitin-conjugating enzyme leads to embryonic le-
thality (7), the Caenorhabditis elegans let-70/ubc-2 ubiquitin-
* Corresponding author. Mailing address: Department of Molecular
Biology, Princeton University, Princeton, NJ 08544. Phone: (609) 258-
2900. Fax: (609) 258-3345. E-mail: email@example.com.
conjugating enzyme is essential for larval development (32),
and mutation of the Drosophila fat facets deubiquitinating en-
zyme 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 embryogen-
esis makes it a candidate for a gene underlying the s1Acrgde-
fect. 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)
Expression analysis. Whole-mount in situ hybridization to embryos was per-
formed as described by Wilkinson and Nieto (30). Digoxigenin-labeled RNA
probes were synthesized with T7 polymerase. The antisense Uch-L3 probe in-
cluded 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 mor-
pholinepropanesulfonic 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 synthe-
sized from fragments of Uch-L3 (wild-type mUCH4 and mutant ?3-7 [Uch-
L3?3-7]) and ?-actin cDNA clones.
Reverse transcription (RT)-PCR was performed with a cDNA cycle kit (In-
vitrogen). Primers used to amplify Uch-L3?3-7RNA were 5?-ATGGAGGGTC
AACGCTGGCT-3? and 5?-GGTGTTTCTGTCAAGATGCTAT-3?. PCR prod-
ucts 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-L3?3-7mutant mice. To delete the 9.5-kb
region encoding exons 3 to 7, two flanking genomic DNA fragments were sub-
cloned into the targeting vector pLOX-PNT, which contains the neomycin re-
sistance gene driven by the phosphoglycerol kinase 2 promoter (PGK-NEO) and
herpes simplex virus thymidine kinase (25). Targeting arms were subcloned from
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-L3?3-7are underlined; the caret denotes conserved cysteine 95.
VOL. 20, 2000ANALYSIS OF MOUSE Uch-L3 2499
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-L3?3-7targeting construct was linearized at a unique NotI site and
electroporated into CJ7 embryonic stem (ES) cells (28), followed by selection
with 125 ?g of active G418 (Sigma) per ml and 1 ?M 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 target-
ing 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 genotyp-
ing was performed on tail DNA with a common forward primer from the
genomic sequence flanking the deletion (5?-GGAACTACTGAGCCATATGTG
C-3?). This primer was used with either a reverse primer derived from endoge-
nous DNA within the deletion for detecting the wild-type allele (5?-CCGACTT
ACTCCATCACTTCAC-3?) or a reverse PGK primer from the NEO cassette for
detecting the targeted allele (5?-CTTGTGTAGCGCCAAGTGC-3?). PCR con-
ditions 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 chromo-
some 13q22. As the s1Acrgregion 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 hu-
man UCH-L3 cDNA cross-hybridized to the BAC contig over
the s1Acrgminimal 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 tran-
script 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% iden-
tity to its mouse paralogue UCH-L1 (Fig. 2).
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 de-
ubiquitinating enzymes (for reviews, see references 4 and 31).
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.
2500 KURIHARA ET AL.MOL. CELL. BIOL.
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 neuro-
degeneration, the likely in vivo function of Uch-L1 is to stim-
ulate protein degradation in neurons where it is primarily ex-
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 s1Acrgphenotype by whole-mount in
situ hybridization (Fig. 3). These include the edges of the open
neural folds, which fail to close in s1Acrgmice, 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 embryo-
To characterize the expression of Uch-L3 in adult mice,
Northern analysis of RNAs isolated from multiple organs was
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.
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).
VOL. 20, 2000 ANALYSIS OF MOUSE Uch-L3 2501
performed (Fig. 4). The Uch-L3 transcript was ?900 nucleo-
tides 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 in-
trathymic T-cell differentiation, both of which are dependent
on the ubiquitin pathway.
Generation and analysis of Uch-L3?3-7mice. 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
FIG. 5. Construction of a targeted Uch-L3?3-7allele. (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-L3?3-7allele 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-L3?3-7(?/?) testes were hybridized to Uch-L3 and ?-actin probes.
2502 KURIHARA ET AL.MOL. CELL. BIOL.
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.
Since only a portion of Uch-L3 could be targeted due to its
large size, it was most critical to remove residue 95, the cata-
lytic 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 s1Acrgphenotype is recessive, Uch-L3?3-7het-
erozygotes were bred to homozygosity. Mice homozygous for
Uch-L3?3-7were obtained at weaning at the expected Mende-
lian frequency. To assess the transcripts from Uch-L3?3-7,
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-L3?3-7RNA was composed of two prod-
ucts. 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 gener-
ated Uch-L3?3-7/s1Acrgcompound 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 s1Acrgphenotype, 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-L3?3-7developed 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-L3?3-7ne-
onates 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 embry-
onic tissues. Similarly, although Uch-L3 is expressed in many
adult tissues, no histological defects were observed in hema-
toxylin-eosin-stained sections of mutant kidney, spleen, thy-
mus, 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-L3?3-7homozygotes.
Within the testes, the ubiquitin pathway is required during
spermatogenesis, as shown by the male sterility that is associ-
ated 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 prota-
mines. However, fertility and sperm morphology were unaf-
fected in Uch-L3?3-7homozygous mice (data not shown).
Within the thymus, differentiation of CD4?CD8?T lym-
phocytes is dependent on the generation of major histocom-
patibility complex (MHC) I peptide antigens by the ubiquitin
pathway (for a review, see reference 20). For example, a mu-
tation 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 anti-
gens. However, in Uch-L3?3-7mice, no significant reduction in
CD4?CD8?T lymphocytes within the thymus or spleen was
observed by FACS analysis with CD4, CD8, and T-cell recep-
tor ?? antisera (Table 1). In addition, MHC I cell surface
staining was not significantly reduced in Uch-L3?3-7mice when
assayed by FACS analysis with H-2Kbantiserum (Table 1).
The absence of either an embryonic or an adult phenotype in
Uch-L3?3-7mice implies either that Uch-L3 performs an un-
detected nonessential function or that Uch-L3 is functionally
redundant with Uch-L1. Uch-L3 and Uch-L1 display 52% iden-
tity, 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 pat-
terns have not been demonstrated, such as during embryogen-
esis, 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 deubiquiti-
nating enzymes, it is expected that they possess distinct sub-
strate specificities. Experiments are under way to test these
We are grateful to Robert S. Ingram for sequencing of cDNA clones
and to Audrey Goddard at Genentech, Inc., for genomic DNA se-
quencing. 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
L.J.K. was supported by an NRSA award from the National Insti-
tutes of Health. S.M.T. is an investigator of the Howard Hughes
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TABLE 1. T-cell differentiation and MHC I expression
in Uch-L3 mutant mice
% of CD4?CD8?
Surface MHC I staining
7.98 ? 2.28
8.89 ? 3.19
48.31 ? 2.5
46.09 ? 1.15
7.85 ? 1.20
6.50 ? 1.43
47.45 ? 2.7
49.71 ? 4.26
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