Download Isoform-Specific Mutations in the Caenorhabditis elegans

Copyright  2001 by the Genetics Society of America
Isoform-Specific Mutations in the Caenorhabditis elegans Heterochronic Gene
lin-14 Affect Stage-Specific Patterning
Brenda J. Reinhart and Gary Ruvkun
Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics,
Harvard Medical School, Boston, Massachusetts 02114
Manuscript received June 19, 2000
Accepted for publication September 20, 2000
ABSTRACT
The Caenorhabditis elegans heterochronic gene lin-14 specifies the temporal sequence of postembryonic
developmental events. lin-14, which encodes differentially spliced LIN-14A and LIN-14B1/B2 protein
isoforms, acts at distinct times during the first larval stage to specify first and second larval stage-specific
cell lineages. Proposed models for the molecular basis of these two lin-14 gene activities have included
the production of functionally distinct isoforms and the generation of a temporal gradient of LIN-14
protein. We report here that loss of the LIN-14B1/B2 isoforms alone affects one of the two lin-14 temporal
patterning functions, the specification of second larval stage lineages. A temporal expression difference
between LIN-14A and LIN-14B1/B2 is not responsible for the stage-specific phenotype: protein levels of
all LIN-14 isoforms are high in early first larval stage animals and decrease during the first larval stage.
However, LIN-14A can partially substitute for LIN-14B1/B2 when expressed at a higher-than-normal level
in the late L1 stage. These data indicate that LIN-14B1/B2 isoforms do not provide a distinct function
of the lin-14 locus in developmental timing but rather may contribute to an overall level of LIN-14 protein
that is the critical determinant of temporal cell fate.
T
HE spatial and temporal patterns of cell division,
morphogenesis, and differentiation are tightly regulated in multicellular organisms. Heterochronic mutations that specifically affect the coordination of the timing or sequence of developmental events (Gould 1977;
Ambros and Horvitz 1984) have been identified in
Drosophila (Ebens et al. 1993), slime mold (Simon et
al. 1992), and plants (Poethig 1988; Lawson and Poethig 1995), but the most extensive analysis of the control of developmental timing has been in the nematode
Caenorhabditis elegans. In C. elegans, a regulatory hierarchy of heterochronic genes has been identified that
coordinately controls the temporal identities of diverse
types of postembryonic cells (Ambros and Moss 1994;
Slack and Ruvkun 1997). The timing of a variety of
developmental decisions are under the control of the
heterochronic genes, including stage-specific cell lineage patterns (Ambros and Horvitz 1984), terminal
differentiation of cell types (Ambros 1989), synaptic
remodeling (Hallam and Jin 1998), dauer larva initiation (Liu and Ambros 1989), and cell cycle progression
(Euling and Ambros 1996).
A key player in the temporal control of C. elegans development is lin-14. Wild-type animals develop through
four larval stages (L1, L2, L3, and L4) followed by the
Corresponding author: Gary Ruvkun, Department of Molecular Biology, Wellman Building, 8th Floor, 50 Blossom St., Massachusetts General Hospital, Boston, MA 02114.
E-mail: [email protected]
Genetics 157: 199–209 ( January 2001)
adult reproductive stage. Null mutations in lin-14 cause
a failure to execute L1-specific fates in many tissues,
which instead prematurely execute L2-specific fates followed by each subsequent stage-specific event occurring
one stage precociously relative to wild type. A study of
partial loss-of-function (lf) mutations has shown that
the lin-14 locus has two independently mutable stagespecific functions, lin-14a and lin-14b, and temperature
shift studies showed that they each act at different times
during development to control stage-specific cell fate
decisions in the V cell lineage (Ambros and Horvitz
1987). lin-14a is required during the early L1 stage to
specify L1 stage cell lineages instead of precocious L2
stage cell lineages, and lin-14b is required later during
the L1 stage to specify L2 stage cell lineages after the
next larval molt instead of precocious L3 stage cell lineages (Figure 1A). These two classes of lin-14 alleles
complement each other, suggesting that the two activities are independent functions of lin-14 (Ambros and
Horvitz 1987).
lin-14b is not required for L2 fate determination, because L2 stage cell lineages are executed in lin-14(null)
mutants, albeit one stage precociously (Ambros and
Horvitz 1984). lin-28, which encodes a protein with
two potential RNA-binding domains (Moss et al. 1997),
is necessary for L2 stage-specific cell lineages (Ambros
and Horvitz 1984). There is a positive regulatory feedback loop between lin-14 and lin-28. LIN-28 is required
to maintain LIN-14 expression late in the L1 stage
(Arasu et al. 1991) and LIN-14 is required sometime
during or after the L1 stage to maintain late expression
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B. J. Reinhart and G. Ruvkun
of LIN-28 in a lin-4(lf) animal (Moss et al. 1997). One
possibility is that the function of lin-14b activity is to fine
tune LIN-28 expression and, therefore, the timing of
L2 fate determination. This lin-14-lin-28 regulatory loop
is not required for L1 fate determination by lin-14a,
since L1 fates occur normally in both lin-28 null mutants
and lin-14b mutants.
Several molecular models could account for the existence of the two lin-14 genetic activities. lin-14 may encode two functionally distinct domains, either in the
same protein product (Klein and Meyer 1993) or in
different isoforms generated by alternative splicing or
alternative promoters (Talbot et al. 1993; Javier Lopez
1995; Ayoubi and van de Ven 1996). The lin-14 locus
uses two promoters to produce three novel proteins by
alternative splicing (Wightman et al. 1991). However,
LIN-14 is also expressed in a temporal gradient. Like the
spatial morphogen gradients of the Drosophila embryo
(Driever and Nüsslein-Volhard 1988; NüssleinVolhard 1991), different levels of LIN-14 protein
across time could direct developmental stage-specific
cell fates. The overall LIN-14 level is highest in the early
L1 stage and decreases during the L1 stage until it is
almost absent in the L2 and later stages of development
(Ruvkun and Giusto 1989). This downregulation is
mediated by lin-4, which encodes a regulatory RNA complementary to multiple sequence elements in the 3⬘
untranslated region (UTR) of all lin-14 transcripts and
negatively regulates translation of the lin-14 mRNA (Lee
et al. 1993; Wightman et al. 1993). Either gain-of-function (gf) lin-14 mutations or lin-4(lf) mutations cause
an inappropriately high LIN-14 level in late development and result in the reiteration of L1 or L2 larval
lineages (Ambros and Horvitz 1984; Ruvkun and
Giusto 1989; Arasu et al. 1991).
To test these models of the lin-14a and lin-14b genetic
functions, we molecularly characterized a number of
lin-14 mutations, including four lin-14b alleles, and examined the LIN-14 isoforms for stage-specific expression. We find that the loss of lin-14b activity alone is
due to mutations in an exon common to the LIN-14B
isoforms, including a nonsense mutation that is a probable LIN-14B1/B2 null allele. Thus the lin-14b mutant
phenotype results from a specific loss of the LIN-14B1/
B2 isoforms, and expression of LIN-14A only is sufficient
to specify L1 but not L2 fates. We show that the temporal
regulation of LIN-14A and LIN-14B1/B2 isoform expression is similar and that LIN-14 levels are decreased
throughout the animal in a lin-14b null mutant. However, an increase in LIN-14A protein induced by a null
mutation in the lin-4 negative regulatory RNA can partially substitute for a loss of LIN-14B activity in the specification of L2 patterns of cell lineage. These data suggest
that the major LIN-14 isoforms are functionally similar
and that it is the decrease in the sum total of LIN-14
isoform abundances in the lin-14b mutant that causes
temporal patterning defects. Interspecies comparison
of lin-14 from the related species C. briggsae reveals that
the complex structure of this locus is conserved with all
three splice forms present, favoring a functional importance for these isoforms in the generation of graded
LIN-14 expression. These data suggest complex regulation of the accumulation and graded decline in LIN-14
protein abundance to temporally pattern the C. elegans
postembryonic cell lineage.
MATERIALS AND METHODS
Strains: All experiments were performed at 20⬚. lin-14 and
lin-4 double mutants were constructed as follows: MT1155
lin-4(e912)/mnC1; him-5(e1467ts) males were mated to lin14(n536n540)/szT1 hermaphrodites, and cross-progeny lin4(e912)/⫹; him-5(e1467ts)/⫹; lin-14(n536n540)/⫹ were identified by placing a single F1 animal on a plate and checking
for the Lin phenotypes of both lin-4 and lin-14 in the F2 progeny. F2’s with the lin-4(e912) phenotype were singled and lin4(e912); lin-14(n536n540) was identified in the F3 progeny by
the suppression of the lin-4(e912) phenotype. For lin-4(e912);
lin-14(n355n534), MT1155 males were mated to lin-14(n355
n534) hermaphrodites that were cured for the lin-14 vulval
defect post-dauer formation (Liu and Ambros 1991), nonLin F1 hermaphrodites lin-4(e912)/⫹; him-5(e1467ts)/⫹; lin14(n355n534)/⫹ were singled, and the homozygous double
mutant was identified in the F3 by the suppression of the lin4(e912) phenotype. Since the strains display a suppression of
the lin-4(e912) phenotype, we verified the genotype by Southern analysis as previously described (Lee et al. 1993).
Molecular characterization of lin-14 alleles: For Southern
analysis of the lin-14 alleles, genomic DNA was digested with
HindIII and probed with the following lin-14 fragments: cosmid KKH9 (promoter region); 7.8 kb BglII (promoter just
upstream of exon 1); EcoRI-SacI fragment of pP14B1 (a cDNA
subclone of lin-14B1 containing exons 1, 2, and 3); 6.2 kb
BglII (intron 3); and 7.6 kb BglII (entire lin-14A region and
3⬘ UTR; Ruvkun et al. 1989; Wightman et al. 1991). From 11
alleles determined not to have deletions outside the 3⬘ UTR
by Southern analysis, coding regions and splice junctions were
amplified by PCR from genomic DNA, and the resulting product was directly sequenced (Promega, Madison, WI). Both
DNA strands and a second PCR reaction were sequenced to
verify mutations. We have described the location of mutations
relative to nucleotide positions in C. elegans genomic cosmid
clone T25C12 to avoid amino acid numbering changes among
alternatively spliced protein isoforms. All alterations from N2
sequence are reported in results with the exception of an
additional T22933G nucleotide change in intron 8 of lin14(n536n540).
C. briggsae lin-14 analysis: C. briggsae genomic EcoRI subclones previously made from a ␭ library isolate (Ha et al. 1996)
were analyzed by Southern hybridization (Sambrook et al.
1989) using C. elegans lin-14 fragments as probes to identify
subclones containing exons 4–13. Coding regions were sequenced on both strands (USB Sequenase). To obtain the
remainder of the C. briggsae lin-14 sequence, the same library
was probed as previously described (Ha et al. 1996) with a
cDNA fragment containing exons 1, 2, and 3. All six positive
clones had identical inserts. HindIII fragments of the insert
were subcloned into pBluescriptSK (Stratagene, La Jolla, CA)
and coding regions were sequenced on both strands (USB
Sequenase). For RT-PCR analysis of splice variants, total RNA
was prepared from C. briggsae, and first strand cDNA was made
using a lin-14-specific primer (R4) and SuperScript RNase
H-Reverse Transcriptase according to the manufacturer’s in-
Isoform-Specific lin-14 Mutations
structions (GIBCO-BRL, Gaithersburg, MD). PCR was done
with primer pairs in exons 1 and 5 or exons 4 and 5 and the
resulting products were analyzed by electrophoresis on 2%
agarose gels.
Isolation and purification of LIN-14A and LIN-14B antibodies: PCR fragments containing either exon 3 or exon 4 of lin14 were generated and subcloned in frame downstream of
glutathione S-transferase (GST) in pGEX-2T (Smith and
Johnson 1988) using BamHI and EcoRI sites in the primers.
Fusion proteins were induced in the host strain DH5␣. Soluble
GST-exon 3 fusion protein was affinity purified on glutathione
Sepharose 4B (Pharmacia, Piscataway, NJ) and used to immunize rabbits (Charles River Pharmservices). The GST-exon 4
fusion protein is insoluble. Protein was obtained from inclusion bodies, further purified on a 12% polyacrylamide gel,
and the specific protein band was excised for injection into
rabbits. Primary injections with 250 ␮g of protein per animal
were followed by boosts every 3 weeks with 100 ␮g of protein.
Antibodies specific to LIN-14 were detected by Western blotting after three boosts.
Anti-LIN-14B1 and B2 antibodies were purified with His6:
LIN-14B1, and anti-LIN-14A antibodies were purified with
His6:LIN-14A. Affinity columns were prepared by coupling
purified His6 fusion proteins (QIAGEN, Valencia, CA) to
CNBr-activated Sepharose 4B (Pharmacia), and antibodies
were purified by standard methods (Harlow and Lane 1988).
The resulting purified antibodies were tested on Western blots
(as described below) using in vitro-produced proteins from all
three full-length cDNA sublones to verify specific reactivity
with the N-terminal exons.
Western analysis: Bulk embryos were collected by treatment
of mixed-stage animals with 25% Clorox bleach/0.25 m NaOH
for 5 min and three washes in M9 buffer. Larvae were synchronized as hatchlings by incubating embyros for 18 hr at 20⬚ in S
medium. Postembryonic larval stages were obtained by plating
hatchlings on Escherichia coli OP50 as a food source and harvesting at the appropriate timepoint after the initiation of feeding.
Developmental hallmarks (Sulston and Horvitz 1977) were
observed to monitor proper progression of growth.
Protein from worms was extracted by boiling for 10 min in
2% SDS, 50 mm Tris-Cl (pH 6.8), and 10% glycerol, and total
protein in the lysate was quantitated using the Biorad DC
protein assay. Before loading, 100 mm dithiothreitol and 0.1%
bromophenol blue were added to the samples, which were
boiled again for 3 min. Samples were run on 7.5% SDS-polyacrylamide gels and electroblotted to enhanced chemiluminescence (ECL) nitrocellulose (Amersham, Buckinghamshire, UK; Sambrook et al. 1989). In Western analysis, 2%
ovalbumin was used as a blocking agent for anti-LIN-14A, 1%
BSA/4% nonfat powdered milk was used for anti-LIN-14 C
terminus and anti-LIN-14B1/B2. For E7 anti-␤ tubulin mouse
monoclonal (Developmental Studies Hybridoma Bank, University of Iowa), 5% nonfat powdered milk was used for the
initial blocking, and 0.5% BSA was used in antibody incubations. Primary incubations were overnight at 4⬚ in Tris-buffered saline/Tween 20 (TBST; 150 mm NaCl, 10 mm Tris pH
7.5, 0.1% Tween) plus blocking agent with either anti-LIN-14
C-terminal antibody (1:1000; Ruvkun et al. 1989), anti-LIN14B (1:500), anti-LIN-14A (1:250), or E7 (1:250), and secondary incubations were 2 hr at room temperature with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody
(Amersham) diluted 1:2500 in TBST plus blocking agent.
Bands were detected using ECL (Amersham).
Indirect immunofluorescence: Whole-mount fixation of animals was performed according to a modification of the method
of Finney and Ruvkun (Epstein and Shakes 1995) with fixation in 1% paraformaldehyde for 30 min at 4⬚. Primary antibody dilutions were 1:50 anti-LIN-14 C terminus (Ruvkun and
201
Giusto 1989), 1:1500 MH27 (Francis and Waterston 1991),
or 1:75 anti-MEC-7 (Hamelin et al. 1992). Our exon-specific
sera did not react strongly with their antigens under any conditions tested unless the proteins were highly overexpressed on
transgenic arrays and, therefore, they were not used as primary
antibodies for our studies. Secondary antibodies were 1:100
FITC-conjugated goat anti-rabbit antibody ( Jackson ImmunoResearch, West Grove, PA) for anti-LIN-14 and anti-MEC-7
or 1:100 rhodamine-conjugated goat anti-mouse for MH27.
Mixed-stage samples were double stained with anti-LIN-14 and
MH27. The developmental stage of animals was determined
by gonadal progression (Hirsh et al. 1976) seen by 4⬘,6-diamidino-2-phenylindole (DAPI) staining as well as lateral hypodermal and vulvul development judged by MH27 staining. Control stainings of the samples to verify equal fixations were
done with anti-MEC-7. Animals were mounted on 2% agarose
pads in Vectastain mounting medium (Vector Labs, Burlingame, CA) with 0.1 mg/ml DAPI. Photographs were taken
with a digital imaging system and compiled in Adobe Photoshop.
Timing of the terminal differentiation of the lateral hypodermis: To observe the extent of alae formation in larval and
adult stages, living animals were anesthetized with sodium
azide and observed using Nomarski optics. Fourth larval stage
animals and young adults were identified by progress of the
animal’s gondal development (Hirsh et al. 1976), which is not
affected by mutations in lin-14 or lin-4 (Ambros and Horvitz
1984). The extent of alae formation was judged on a single
side of each animal. To count V cells, mixed-stage populations
of animals were fixed and stained with the MH27 antibody as
described above.
RESULTS
Molecular analysis of lin-14 alleles: We identified the
molecular nature of mutations in lin-14 alleles previously analyzed for their effects on temporal cell fates in
the lateral hypodermal lineages (Ambros and Horvitz
1987). We were particularly interested in the four partial
loss-of-function lin-14b alleles that have L2 stage-specific
defects. These mutant animals have normal L1 stage
development in the V cells of the lateral hypodermis,
unlike complete loss-of-function lin-14 alleles, but they
lack L2 stage-specific cell divisions and proceed to L3
patterns of cell division directly from L1 (Figure 1A).
Three lin-14 transcripts are generated from two promoters (Wightman 1992 and Figure 1B). The 5⬘ ends
of two classes of transcripts, lin-14A and lin-14B1/B2,
are separated in the genome by almost 15 kb. The lin14B transcripts are differentially spliced to generate the
LIN-14B1/B2 isoforms, which splice two or three exons,
respectively, over a 12-kb intron to a set of nine exons
common to lin-14A and lin-14B1/B2. The LIN-14A isoform uses a promoter within the lin-14B1/B2 transcription unit to transcribe a unique exon 4 that splices to
these nine common exons (Wightman 1992).
All four lin-14b mutant alleles had molecular lesions
predicted to affect only the LIN-14B1 and B2 proteins
(Figure 1B, Figure 2, and Table 1). Two are predicted
to produce neither LIN-14B protein. The EMS-induced
allele n534, a revertant of the n355 gain-of-function
allele, is an amber mutation in the third exon that is
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B. J. Reinhart and G. Ruvkun
Figure 1.—Molecular lesions associated with
lin-14 alleles. (A) Phenotypes of lin-14 loss-of-function alleles in the lateral hypodermal cells,
adapted from Ambros and Horvitz (1987).
Three horizontal lines indicate the adult cell fate,
which is cessation of cell division and lateral adult
alae formation on the cuticle. lin-14(a-b-) and lin14(b-) hermaphrodites undergo a wild-type number of molts but are lacking V-cell divisions at
the time of the fourth larval molt. (B) Mutations
specific to the LIN-14B1/B2 isoforms result in the
loss of lin-14b genetic activity. Point mutations
associated with lin-14 alleles are indicated above
the genomic DNA; genomic rearrangements and
deletions are indicated below. Open reading
frames are shown in black, 5⬘ untranslated regions
in exons 1 and 4 and the 3⬘ untranslated region
in exon 13 are in white, and exons are numbered
at the bottom of the diagram. Double diagonal
lines indicate an area not drawn to scale. Locations of the lin-14(n360) and lin-14(n407) breakpoints were approximated as described in materials and methods. Locations of the lin-14(n355),
lin-14(n536), and lin-14(n536n838) lesions were
originally reported in Wightman et al. (1991).
predicted to truncate prematurely both LIN-14B1 and
B2 but leave LIN-14A unaffected, because exon 3 is not
spliced into the lin-14A transcript. n360 is a gamma-rayinduced allele previously reported to have polymorphisms near exon 3 and upstream in the promoter,
suggesting a possible inversion (Ruvkun et al. 1989).
Exon 3 is rearranged such that it cannot be amplified
by PCR using exon-specific primers, but the first two
exons appear to be unaffected (see materials and
methods, data not shown). The other two EMS alleles,
n727 and n840, both substitute tyrosine for cysteine at
the same postition (Figure 1B, Figure 2, and Table 1).
The functional importance of this residue is unknown,
but it is conserved among three Caenorhabditae. We are
certain that this substitution occurred independently,
because n840 was isolated as a revertant of n355 and
the n355 3⬘ UTR rearrangement can be detected in
lin-14(n355n840) genomic DNA but not in lin-14(n727)
DNA (data not shown).
Alleles that decrease both lin-14a and lin-14b genetic
activities are caused by mutations in the region common
to all isoforms (Figure 1B, Figure 2, and Table 1). The
majority were isolated as revertants of the gain-of-func-
tion alleles n355 and n536 (Ambros and Horvitz 1987
and Table 1). n540, which gives the most severe lossof-function phenotype and behaves as a genetic null
(Ambros and Horvitz 1987), is an amber mutation in
the eighth exon that is predicted to truncate almost
50% of the coding sequence from all three proteins.
Five other mutations affect both genetic activities but
are not complete genetic nulls. Three are substitutions
(n539, n179, and n530) and a fourth is a splice donor
mutation (n531). The fifth, n407, is a gamma allele with
a polymorphism near exon 3 (Ruvkun et al. 1989) but
the nature of the alteration is unclear. The rearrangement
does not appear to be a simple deletion by Southern
analysis (see materials and methods), suggesting either an insertion or an inversion with a breakpoint far
outside the coding region (data not shown).
lin-14(n536n538) and lin-14(n355n679ts) animals have
complex phenotypes. In the lateral hypodermis of these
mutants, some cells have gain-of-function phenotypes
and some have loss-of-function phenotypes, presumably
due to only partial reversion of the dominant mutant
phenotype (Ambros and Horvitz 1987). Both are substitutions in the common region. The revertant lesion
Isoform-Specific lin-14 Mutations
203
Figure 2.—Protein sequence comparison between C. elegans, C. briggsae, and C. vulgaris LIN-14. Genomic C. briggsae clones
(AF304859–AF304863) were identified and sequenced as described in materials and methods, and the compiled protein
sequence is shown here. One C. vulgaris lin-14 partial cDNA (no. AAF34229) has been reported previously (Hong et al. 2000).
The first three lines are alternatively spliced exons present in the isoforms indicated below the exon number. LIN-14B2 is
predicted to start at the first methionine in exon 3 (the fourth amino acid listed). Exons 5–13 are common to all three LIN-14
isoforms (CAA42791–CAA42793). Amino acid identity is boxed in black, and dots indicate absence of amino acids. Changes in
point mutations are indicated with an arrow above the sequence. Black horizontal lines above sequence in exons 9 and 11
indicate predicted nuclear localization domains (Hong et al. 2000), and white lines underlining sequence in exon 11 indicate
an amphipathic helix (Wightman et al. 1991).
associated with lin-14(n536n838), the only mutation
known to specifically affect lin-14a activity (Ambros and
Horvitz 1987), has been previously reported to be a
point mutation in exon 9 that is expected to affect all
isoforms (Wightman et al. 1991). No mutations specific
to the LIN-14A isoform were found.
Conservation of LIN-14 isoforms in C. briggsae: We
used interspecies comparison between Caenorhabditae
to identify evolutionarily conserved splice forms and
amino acid residues likely to be essential to the role of
the novel LIN-14 protein (Figure 2). Genomic clones
from C. briggsae were identified by hybridization to a C.
elegans lin-14 probe and sequenced (see materials and
methods). The exons specific to the LIN-14B isoforms,
exons 2 and 3, have 86 and 80% amino acid identity
between species, respectively. A C. vulgaris partial cDNA
has been previously identified (Hong et al. 2000). Although the N terminus is incomplete, this cDNA contains a complete exon 3 and the common domain. Exon
3 is 77% identical across all three species, with a potential start codon for the LIN-14B2 product and the sites
of the lin-14b missense mutations all conserved. Since
only a single cDNA was isolated from C. vulgaris, the
extent of conservation of the LIN-14A-specific exon 4
across all three species could not be determined. However, exon 4 is 61% identical between C. elegans and C.
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B. J. Reinhart and G. Ruvkun
TABLE 1
Phenotypic classes of lin-14 alleles and products altered
Genotypea
Mutagenb
Molecular lesion
L1 and/or L2 lineages reiterated
3⬘ UTR rearrangement (3⬘R)
3⬘ UTR internal deletion (3⬘⌬)
n355gf
n536gf
␥-Ray
EMS
n536n540
n536n539
n355n407
n355n531
n179ts
n530ts
EMS/EMS
EMS/EMS
␥-Ray/␥-ray
␥-Ray/EMS
spo
EMS
L1 lineages absent and L2 precocious
3⬘⌬, A22900T (K → amber)
3⬘R, C23269T (L → F)
3⬘R, intron 3 rearrangement
3⬘R, intron 5 splice donor GT → AT
A23013G (R → G)
C23269T (L → F)
n360
n727
n355n534
n355n840
␥-Ray
EMS
␥-Ray/EMS
␥-Ray/EMS
L2 lineages absent
Exon 3 rearrangement
G9229A (C → Y)
3⬘R, G9190A (W → amber)
3⬘R, G9229A (C → Y)
n536n838
EMS/EMS
L1 lineages absent
3⬘⌬, G23070A (A → T)
n536n538
n355n679ts
EMS/EMS
␥-Ray/EMS
Complex
3⬘⌬, C23323T (P → S)
3⬘R, T23002A (V → D)
Products affected
All
All
LIN-14B1 and B2
All
All
Phenotypic classes were determined by Ambros and Horvitz (1987); L1, first larval stage; L2, second larval
stage; complex phenotypes have some lineages reiterated, others absent. Allele changes for n355gf, n536gf,
and n536n838 were originally described by Wightman et al. (1991). Positions of lesions are given in the context
of genomic cosmid T25C12 to avoid numbering changes among the alternatively spliced protein isoforms.
a
Genotypes with two allele numbers refer to loss-of-function (lf ) alleles obtained by reversion of a linked
gain-of-function (gf ) allele; ts, temperature sensitive.
b
Mutagen used in the isolation of the allele; EMS, ethyl methanesulfonate; spo, spontaneous; mutagens for
a gf allele and its lf revertant are listed in the order of gf/lf.
briggsae, perhaps suggesting a lesser degree of conservation than that of the LIN-14B specific exons. All three
splice forms of lin-14 are present in C. briggsae as determined by reverse transcription of total RNA and PCR
using exon-specific primers (data not shown).
In the region common to all three LIN-14 isoforms,
the highest percent amino acid identity among all three
species is in exons 9, 10, and 11, which are 93% identical.
Loss-of-function mutations cluster in this region; the
null allele lin-14(n536n540) truncates the protein before
exon 9, and all point mutations in the common region
fall in exons 9 and 10. A predicted amphipathic helix
in exon 11 (Wightman et al. 1991) and two potential
consensus sequences for nuclear localization in exons
8 and 11 (Hong et al. 2000) are 100% identical but we
have observed no point mutations within these regions.
The most highly diverged section of the protein coding
region is the C terminus, where conservation across all
three species quickly falls off in exons 12 and 13, although the C. briggsae and C. vulgaris proteins remain
57% identical.
Expression analysis of the LIN-14 isoforms: Previous
investigations of LIN-14 expression have examined the
stage-specific regulation of the LIN-14 proteins using
an antibody that recognizes all three isoforms (Ruvkun
and Giusto 1989). These analyses showed temporal
downregulation of LIN-14 during the L1 stage, when
expression of LIN-14 is negatively regulated by the small
RNA lin-4. lin-4 RNA is expressed weakly at 12 hr of
postembryonic development and increases to high levels
by 16 hr, late in the L1 stage (Feinbaum and Ambros
1999). Expression differences among the individual isoforms in mutant backgrounds or stages of development
would not have been seen. To analyze expression of
the isoforms individually, we prepared isoform-specific
antibodies that recognize either exon 3 (common to
LIN-14B1 and B2) or exon 4 (unique to LIN-14A) and
verified their specific reactivity with these exons by Western analysis using in vitro translated proteins (see materials and methods).
One possible explanation for the inability of LIN-14A
protein to direct L2 fates in the lateral hypodermis of
lin-14b mutants is that LIN-14A is not expressed in the
late L1 stage, the time at which L2 fate decisions are
being made in this tissue (Ambros and Horvitz 1987).
However, developmental Westerns showed little difference in the overall temporal regulation of the LIN-14
isoforms in wild-type animals. Both LIN-14A and LIN-
Isoform-Specific lin-14 Mutations
14B1/B2 protein levels are high at the early L1 stage,
begin to decrease by 6–9 hr of postembryonic development, and steadily decrease throughout the remainder
of the first larval stage to almost undetectable levels by
early L2 (Figure 3A). These data suggest that either the
loss of LIN-14B1/B2 in the lin-14b mutants lowers the
overall level of functional LIN-14 protein or that temporal differences in LIN-14 isoform expression are tissue
specific and not detectable by Western analysis.
To distinguish between these possibilities, we examined LIN-14A expression in a LIN-14B1/B2 null mutant
using immunofluorescence analysis. First, we verified
our prediction that lin-14(n360) and lin-14(n355n534)
are LIN-14B1/B2 molecular nulls by Western analysis
of protein extracts from the lin-14b mutants. The LIN14B1/B2 proteins, which comigrate near 67 kD, were
detected in wild-type C. elegans as well as in strains bearing either of two LIN-14B missense alleles, lin-14(n727)
and lin-14(n355n840), but were absent in lin-14(n360)
and lin-14(n355n534) (Figure 3B). We frequently observed reduced levels of LIN-14A protein in lin-14(n355
n534) and lin-14(n355n840) hatchlings by Western analysis or immunostaining as compared to lin-14(n360) and
lin-14(n727) (data not shown, Wightman et al. 1991),
which have analogous loss-of-function lesions (see
above). The simplest explanation is that another mutation lies outside the coding region and was not detected
in our analysis. However, both backgrounds with varying
protein levels are revertants of the gain-of-function allele lin-14(n355). While lin-14(n355) animals have no
obvious LIN-14 instability, the n355 3⬘ UTR rearrangement does remove several blocks of conserved sequence
with unknown function, and these could serve as positive
regulatory elements under certain conditions.
Because of this inconsistency of LIN-14 expression in
lin-14(n355n534), we focused our study on lin-14(n360).
Only LIN-14A protein remains in lin-14(n360) (Figure
3B). By immunofluorescence analysis using an antibody
to the C terminus of LIN-14, total LIN-14 protein in lin14(n360) animals is less abundant in the early L1 stage
than in wild-type animals (Figure 4A). Expression was
not missing from any tissue type that normally expresses
LIN-14. While levels of LIN-14 are only beginning to
decrease in early to mid-L1 stage wild-type animals at 5–6
hr of postembryonic development, lin-14(n360) animals
often have much lower levels of LIN-14 (Figure 4A).
One possibility is that the overall level of LIN-14 is decreased in lin-14(n360) such that LIN-14 levels fall below
the threshold for L2 fate determination but are sufficient for L1 fate determination.
LIN-14A is sufficient for lin-14a and lin-14b function:
Although LIN-14A is present in lin-14b mutants at the
late L1 stage, when the lin-14b temperature-sensitive
period shows that L2 fate decisions are specified
(Ambros and Horvitz 1987), it is either unable to
influence L2 fates or it is expressed at an insufficient
level to do so in lin-14b mutants. To test between these
205
Figure 3.—Western analysis of LIN-14 isoform temporal
regulation. Approximately 2 ␮g of total protein was loaded
in each lane. (A) The LIN-14 isoforms do not display stagespecific expression differences. Both exon-specific antibodies
detect a steady decrease of protein from hatching through the
L1 larval stage. Animals were staged as described in materials
and methods, and the L1 molt occurs at ⵑ15 hr of postembryonic development. Note the doublet observed with anti-LIN14B, which may represent the two LIN-14B splice variants, B1
and B2. (B) lin-14(n360) and lin-14(n355n534) are null mutants for the expression of the LIN-14B1 and B2 proteins.
Anti-LIN-14B antibody detects protein in extracts from wildtype animals but not in extracts from lin-14(n536n540) animals, which contain an amber mutation affecting all three
predicted LIN-14 proteins, or from lin-14(n360) or lin14(n355n534) animals, which were both expected to be molecular nulls for LIN-14B. Anti-C-terminal antibody did not detect
altered size proteins in lin-14(n360) or lin-14(n355n534)
extracts that would have suggested use of an alternative translational start site downstream of the mutant lesions. Anti-LIN14A antibody detects protein in extracts from all mutant backgrounds tested except for the LIN-14 nonsense mutant
lin-14(n536n540). No truncated proteins were detected in lin14(n536n540) extracts with any of the three anti-LIN-14 antibodies, suggesting that the altered transcripts were degraded
by the nonsense-mediated decay mechanism of C. elegans.
206
B. J. Reinhart and G. Ruvkun
Figure 4.—LIN-14 expression analysis. Animals were co-immunostained
with antibodies to the common domain
of the nuclear LIN-14 proteins (shown)
as well as the monoclonal antibody
MH27 (data not shown) to outline hypodermal cells for developmental stage
determination. (A) LIN-14 expression is
reduced in the LIN-14B null mutant lin14(n360) by the mid-L1 stage, but the
level of LIN-14 expression can be increased to almost wild-type levels in the
double mutant lin-4(e912); lin-14(n360).
(B) LIN-14A temporal misexpression
is not maintained in lin-4(e912); lin14(n360) animals. LIN-14 is barely detectable in a wild-type L2 stage animal
(Ruvkun and Giusto 1989) although
a lin-4(e912) L2 stage animal shows a
high level of LIN-14 accumulation.
lin-4(e912); lin-14(n360) animals accumulate slightly more LIN-14 than wild
type but far less than lin-4(e912) alone.
possibilities, we increased the level of LIN-14A protein
in a lin-14b mutant background by removing the negative regulator lin-4. In lin-4(e912); lin-14(n360) early- to
mid-L1- stage animals, the level of LIN-14A is increased
significantly as compared to lin-14(n360) animals (Figure 4A).
We examined hypodermal fates in lin-4(e912); lin14(n360) animals to determine whether this increased
level of LIN-14A at the late L1 stage could be sufficient
to restore a wild-type sequence of development in the
lateral hypodermal V cells. As shown in Figure 1A, the
V cells of wild-type animals divide at each larval molt,
with L2 stage-specific double divisions of V1–V4 and V6
after the L1-to-L2 stage molt. At the L4-to-adult stage
molt, these cells cease division and fuse with the other
seam cells, H0, H1, H2, and T, to form bilateral syncytia
that contribute to the synthesis of adult specific cuticle
structures called alae. The alae appear one stage early
at the L3-to-L4 stage molt in lin-14(n360) animals due
to the loss of the L2 stage-specific divisions (Table 2;
Ambros and Horvitz 1987). lin-4(e912); lin-14(n360)
animals show a partial suppression of this mutant phenotype: 50% of L3-to-L4 molt animals exhibit patches of
larval stage cuticle separated by precocious patches of
adult alae (Table 2). This effect requires LIN-14A rather
than another developmental gene regulated by lin-4,
because these patches of dividing seam cells at the L3to-L4 stage molt are not seen in lin-4(e912) animals carrying lin-14(n536n540), a null mutation that is missing
all LIN-14 isoforms (Table 2).
We directly examined the V cells of lin-4(e912); lin-
14(n360) animals to determine whether they executed
a wild-type sequence of development or still deleted L2
stage patterns of cell lineage as in lin-14(n360). On both
the left and the right side of an L1 stage animal, a set
of six V cells lie in a line at the lateral “seam” of the
hypodermis. In wild-type animals entering the L2 stage,
the V1–V4 and V6 cells undergo two rounds of division
to produce four cells from each of these V cells on both
sides of the animal (Figure 5A). Then the number of
V cells is quickly reduced when the anterior daughters
of the second round of division fuse with the hyp7 syncytial cell, resulting in a total of 11 V cells (2 cells each
from the five double divisions of V1–V4 and V6 plus 1
cell from V5) on each side of wild-type L3 animals (n ⫽
10 sides examined). In lin-14(n360) animals, which are
missing the L2-specific V-cell double divisions, a single
division occurs and only two daughter cells are produced from each V cell (Figure 5B), resulting in only
6 V cells on each side of lin-14(n360) L3 animals (n ⫽
6 sides). However, the number of V cells per side of lin4(e912); lin-14(n360) L3 stage animals varied from 6 to
11, with an average of 8 (n ⫽ 9 sides). Figure 5C shows
the V3 cell of an L2 stage lin-4(e912); lin-14(n360) animal
dividing a second time, characteristic of wild-type L2
stage V-cell fate. Consistent with the mosaic fate seen
by patches of adult and larval tissue in the cuticle of lin4(e912); lin-14(n360) animals (Table 2), both L1- and L2type divisions are seen in this single animal: the anterior
daughter of V2 has undergone a single division and the
border of the anterior daughter is fading as it begins
to fuse with the hyp7 cell (Figure 5C, arrowhead), indic-
Isoform-Specific lin-14 Mutations
207
TABLE 2
Lateral hypodermal phenotypes in heterochronic mutants
Genotype
% alae at L4
% alae at adult
Wild type
lin-14(n360)
lin-14(n355n534)
lin-14(n536n540)
lin-4(e912)
lin-4(e912);
lin-14(n360)
lin-4(e912);
lin-14(n355n534)
lin-4(e912);
lin-14(n536n540)
0 (n ⬎ 50)
100 (n ⫽ 14)
100 (n ⫽ 17)
100 (n ⫽ 10)
0 (n ⫽ 10)
50 (n ⫽ 26)
50 patchya
100 (n ⬎ 50)
100 (n ⫽ 15)
100 (n ⫽ 11)
100 (n ⫽ 25)
0 (n ⫽ 10)
93 (n ⫽ 29)
7 patchya
100 (n ⫽ 21)
100 (n ⫽ 8)
100 (n ⫽ 20)
100 (n ⫽ 31)
a
Patchy alae appear when animals are a hybrid of adult and
larval temporal fates; some lateral hypodermal cells terminally
differentiate and form alae as in the adult stage and others
continue to divide as in the larval stages.
ative of a failure to execute a second division and, therefore, lack of rescue of the lin-14(n360) phenotype in
this cell. These data indicate that the elevated level of
LIN-14A in lin-4(e912); lin-14(n360) animals is sufficient
to substitute for the loss of LIN-14B1 and B2 in the
execution of L2 patterns of V-cell division.
Although lin-14(n355n534) animals are insensitive to
lin-4 repression due to the n355 rearrangement and are
null mutants for LIN-14B, they are distinctly different
from lin-4(e912); lin-14(n360) animals and show only
precocious alae, indicative of a lack of L2 lineages in
this background (Table 2). This is most likely due to
the reduced level of protein expression we observed in
revertants of n355 (see above). lin-4(e912); lin-14(n355
n534) animals also do not display larval cuticle patches
at the L3-to-L4 stage molt, further demonstrating that
lin-4(e912) is not acting through another locus to restore
L2 stage-specific divisions to lin-4(e912); lin-14(n360) animals.
lin-4(e912) animals reiterate L1 stage patterns of cell
lineage through the adult stage, keeping larval cuticle
characteristics and never producing adult alae (Chalfie
et al. 1981). This is a result of the continuous expression
of LIN-14 in all postembryonic stages of lin-4(e912) animals (Arasu et al. 1991). However, LIN-14 protein levels
were reduced by early L2 in lin-4(e912); lin-14(n360)
animals (Figure 4B) and almost undetectable in later
larval stages (data not shown), suggesting that a nonlin-4-mediated mechanism is responsible for the downregulation of LIN-14A in these animals. Consistent with
a lack of continuous LIN-14 expression in lin-4(e912);
lin-14(n360) animals (Figure 4B, data not shown), the
extent of larval cuticle patches is reduced in the adult
stage, when 97% of lin-4(e912); lin-14(n360) adult animals display full-length adult alae (Table 2).
Figure 5.—An increased level of LIN-14A is sufficient for
both genetic activities of lin-14. Animals were fixed and stained
with MH27 antibody to visualize the cells of the lateral hypodermis. Lineages indicated were deduced from MH27 staining. (A) A wild-type L2 animal undergoes characteristic V-cell
double divisions in the lateral hypodermis. (B) lin-14(n360)
animals lack the double divisions characteristic of the L2 fate
in the V cells. (C) A lin-4(e912); lin-14(n360) L2 animal expresses a mix of single and double divisions in the lateral
hypodermis, indicating a partial rescue of the lin-14b defect
by misexpression of LIN-14A.
DISCUSSION
The function of the lin-14 isoforms: The lin-14 locus
regulates the timing of L1 stage and L2 stage developmental decisions with two independently mutable activities (Ambros and Horvitz 1987). Our data support
the model that the L2 stage specification function of
lin-14 maps to the LIN-14B1/B2 isoform product: four
lin-14b mutations cause amino acid substitutions or truncation of the LIN-14 B1 and B2 isoforms, and these lin14b mutants fail to specify L2 stage fates. However, an
increase of LIN-14A in an animal missing LIN-14B1/
B2 is partially sufficient to specify L2 stage timing, sug-
208
B. J. Reinhart and G. Ruvkun
gesting that the LIN-14 isoforms do not perform distinct
biochemical functions. Consistent with our results on
L2 stage fate specification in the lateral hypodermis,
Hong and co-workers have recently reported that the
common region of the LIN-14 proteins is also sufficient
for lin-14 function in the timing of vulval precursor cell
divisions as well as adult alae formation when overexpressed on a transgenic array (Hong et al. 2000). The
LIN-14A and LIN-14B1/B2 isoforms display similar temporal expression profiles, making it unlikely that the
activities are independently mutable simply because
they are expressed in separate stages of development.
The model that the LIN-14 isoforms “sum” to form a
functional gradient is more consistent with these results.
The gradient model: In the gradient model for lin14 activity, different concentrations of protein in time
elicit different responses from cells (Ambros and Horvitz 1987). The expression of multiple isoforms would
build up the total amount of LIN-14 protein. When the
lin-4 RNA is expressed in the second half of the L1 stage
(Feinbaum and Ambros 1999), it binds to the 3⬘ UTRs
of the lin-14 mRNAs to block LIN-14 translation, reducing the level of LIN-14 and allowing progression to later
larval development (Lee et al. 1993; Wightman et al.
1993). High levels of LIN-14 at the early L1 stage signal
L1 stage-specific events, low levels by mid-L1 indirectly
signal L2 stage-specific events perhaps through lin-28,
and the absence of protein by the L2 stage signals the
progression to the L3 stage. This model is analogous to
the use of transcription factor gradients in Drosophila
pattern formation where binding sites in target gene
promoters are occupied in a concentration-dependent
fashion, allowing distinct patterns of target gene expression at different concentrations of transcription factors
(Driever et al. 1989; Struhl et al. 1989; Jiang and
Levine 1993). The LIN-14 nuclear protein is novel, but
it may, for example, function as a transcription factor or
an activator of transcription factors that in turn interpret
the gradient.
Several observations are consistent with this gradient
model. Previous studies of the lin-14 gain-of-function
alleles demonstrated that the locus is dose sensitive
(Ambros and Horvitz 1987). We have also observed
that the lin-14(n360) mutation appears to create a dosesensitive phenotype. L2 stage events are missing due to
an overall reduction in the level of LIN-14 from the
loss of LIN-14B1/B2, but the mutant phenotype can be
corrected by elevating the level of the remaining LIN14A in lin-4(e912); lin-14(n360).
One piece of data is difficult to reconcile with the
gradient model. lin-14(n355n534) animals have low levels of LIN-14A protein expression throughout development (data not shown, Wightman et al. 1991). Our
gradient model would predict that the low level of LIN14 in lin-14(n355n534) early L1 animals should be insufficient for L1 fates but sufficient for L2 fates. Instead, L1
fates appear normally and L2 are omitted. One possible
explanation is that the isoforms differ in their efficiency
at performing the stage-specific functions of lin-14. Such
a fine tuning of isoform roles in different developmental
contexts has been seen for homeotic genes of Drosophila (Kuziora 1993; Subramaniam et al. 1994). If LIN14A is more efficient at specifying L1 fates than L2 fates,
low levels of LIN-14A could be sufficient for L1 but
higher levels would be required to direct L2 fates. This
is also one explanation of why our temporal misexpression of LIN-14A in lin-4(e912); lin-14(n360) animals was
not fully penetrant in directing L2 stage-specific fates
in the lateral hypodermis. However, the protein was not
misexpressed at very high levels and also could have
been wavering at a threshold for activity. A full understanding of the functional relevance of the LIN-14 gradient requires a more direct test of the developmental
output from different levels of each LIN-14 isoform as
well as an understanding of the biochemical role of
LIN-14.
Two genes, lin-28 and lin-4, influence the level of LIN14 protein. The LIN-14 expression level is regulated by
LIN-28 in a mutual positive feedback loop, and this
regulatory loop is broken late in the first larval stage
when the expression of both proteins is coordinately
reduced by the negative regulator lin-4 (Wightman et
al. 1993; Moss et al. 1997). lin-28 activity is still needed
to maintain the post-L1 stage misexpression of LIN-14
in lin-4(null) animals (Arasu et al. 1991), suggesting that
although lin-28 is an RNA-binding protein, it does not
simply block lin-4 regulation of lin-14 by binding to the
lin-14 mRNA 3⬘ UTR. This suggests the presence of a
lin-4-independent pathway for LIN-14 translational inhibition or degradation that is blocked by lin-28 function.
Consistent with this, we have observed that LIN-14 expression in lin-4(e912); lin-14(n360) late stage animals is
still reduced in the absence of the lin-4 negative regulator. In addition, the decrease in LIN-14 levels by our
Western analysis appears to be earlier than the reported
onset of lin-4 transcription (Feinbaum and Ambros
1999). One explanation of our data is that the lin-28–
lin-14 feedback loop is broken; if lin-14 activity is not
completely restored to a wild-type level, LIN-28 expression is reduced to the point where it no longer blocks
this lin-4-independent negative regulation of LIN-14.
We thank B. Wightman for suggesting a molecular analysis of the
lin-14b alleles, S. S. Lee, S. Nurrish, and C. Wolkow for comments on
the manuscript, R. Francis and the Waterston laboratory for MH27
antibodies, M. Hamelin and the Chalfie laboratory for MEC-7 antibodies, and the Horvitz laboratory for strains. The E7 ␤-tubulin antibody
was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health
and Human Development and maintained by the University of Iowa,
Department of Biological Sciences, Iowa City, IA 52242. This work
was supported by National Institute of Health grant GM44619 to G.R.
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Communicating editor: P. Anderson