Isoform-Specific Mutations in the Caenorhabditis elegans Heterochronic Gene lin-14 Affect Stage-Specific Patterning

Corresponding author: Gary Ruvkun, Department of Molecular Biology, Wellman Building, 8th Floor, 50 Blossom St., Massachusetts General Hospital, Boston, MA 02114., ruvkun{at}molbio.mgh.harvard.edu (E-mail)

Communicating editor: P. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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.

THE 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 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 stage-specific 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 (Fig 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 ).

View larger version (25K): In this window In a new window Download PPT slide   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 lin-14(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 .

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 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 NUSSLEIN-VOLHARD 1988 ; NUSSLEIN-VOLHARD 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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 lin-14(n536n540)/szT1 hermaphrodites, and cross-progeny lin-4(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 lin-4(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(n355n534) hermaphrodites that were cured for the lin-14 vulval defect post-dauer formation (LIU and AMBROS 1991 ), non-Lin F₁ hermaphrodites lin-4(e912)/+; him-5(e1467ts)/+; lin-14(n355n534)/+ were singled, and the homozygous double mutant was identified in the F₃ by the suppression of the lin-4(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 lin-14(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 instructions (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 lin-14 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 µg of protein per animal were followed by boosts every 3 weeks with 100 µg 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-ß 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-LIN-14B (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 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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 (Fig 1A).

Three lin-14 transcripts are generated from two promoters (WIGHTMAN 1992 and Fig 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 lin-14B 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 (Fig 1B, Fig 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 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-ray-induced 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 (Fig 1B, Fig 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).

View larger version (77K): In this window In a new window Download PPT slide   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 ).

Alleles that decrease both lin-14a and lin-14b genetic activities are caused by mutations in the region common to all isoforms (Fig 1B, Fig 2, and Table 1). The majority were isolated as revertants of the gain-of-function alleles n355 and n536 (AMBROS and HORVITZ 1987 and Table 1). n540, which gives the most severe loss-of-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 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 (Fig 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. 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-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 (Fig 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.

View larger version (40K): In this window In a new window Download PPT slide   Figure 3. Western analysis of LIN-14 isoform temporal regulation. Approximately 2 µg of total protein was loaded in each lane. (A) The LIN-14 isoforms do not display stage-specific 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 15 hr of postembryonic development. Note the doublet observed with anti-LIN-14B, 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 wild-type 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 lin-14(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-LIN-14A 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 lin-14(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.

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 LIN-14B1/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) (Fig 3B). We frequently observed reduced levels of LIN-14A protein in lin-14(n355n534) 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) (Fig 3B). By immunofluorescence analysis using an antibody to the C terminus of LIN-14, total LIN-14 protein in lin-14(n360) animals is less abundant in the early L1 stage than in wild-type animals (Fig 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 (Fig 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.

View larger version (57K): In this window In a new window Download PPT slide   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 lin-14(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); lin-14(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.

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 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 (Fig 4A).

We examined hypodermal fates in lin-4(e912); lin-14(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 Fig 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 L3-to-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 (Fig 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 (Fig 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 lin-4(e912); lin-14(n360) L3 stage animals varied from 6 to 11, with an average of 8 (n = 9 sides). Fig 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 lin-4(e912); lin-14(n360) animals (Table 2), both L1- and L2-type 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 (Fig 5C, arrowhead), indicative 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.

View larger version (62K): In this window In a new window Download PPT slide   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.

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(n355n534) 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 (Fig 4B) and almost undetectable in later larval stages (data not shown), suggesting that a non-lin-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 (Fig 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).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 lin-14b 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, suggesting 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 lin-14 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 dose-sensitive 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 LIN-14A 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 LIN-14 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 LIN-14A 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 LIN-14 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.

	ACKNOWLEDGMENTS

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 ß-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.

Manuscript received June 19, 2000; Accepted for publication September 20, 2000.

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