A. A Temporal Decrease of LIN-14 Protein Controls L1–L3 Events
Genetic analysis of lin-14 has shown that the level of lin-14 activity specifies the expression of stage-specific cell fates for diverse cell types and that a temporal decrease in lin-14 activity is critical for specifying the timing of events during the larval stages (Ambros and Horvitz 1987). Semidominant lin-14(gf) mutations appear to cause an elevated level of lin-14 activity in the L2 when it would normally be reduced and hence result in a reiteration of S1 cell lineage patterns (Ambros and Horvitz 1987). This suggests that lin-14 activity must decrease for the proper expression of L2-specific and later cell fates. Temperature-shift experiments with lin-14 (ts) alleles indicate that lin-14 acts early in the L1 stage to specify the fates of L1 cells and at the end of the L1 stage to control the fates expressed by cells in the early L2. Finally, lin-14 must be absent in the late L2 stage to permit the proper execution of L3-specific fates (Ambros and Horvitz 1987). Thus, lin-14 seems to determine three different sets of stage-specific fates by acting at three successively reduced levels. Hence, the level of lin-14 activity may be the determining factor for temporal cell fate specification in the L1 through L3 stages (Ambros and Horvitz 1987).
The lin-14 gene was cloned by chromosomal walking (Ruvkun et al. 1989). The LIN-14 protein is localized to the nuclei of most of the cells that are affected by lin-14 mutations (Ruvkun and Giusto 1989), consistent with a direct role for lin-14 in the regulation of gene expression. The predicted LIN-14 protein sequence does not indicate homology with previously known proteins, suggesting that lin-14 may represent a new class of regulatory molecule (Wightman et al. 1991). Molecular analysis of lin-14 gene expression confirms that the level of LIN-14 protein is temporally regulated. Western blots and in situ immunofluorescent staining indicated that LIN-14 protein decreases in level between the early L1, when it is abundant, and the end of the L2, when it is undetectable (Ruvkun and Giusto 1989; Wightman et al. 1991). The level of lin-14 mRNA seems to be relatively constant during this time, implying that posttranscriptional regulation is a significant component of the temporal decrease in LIN-14 protein (Wightman et al. 1991, 1993).
Most lin-14 mutations, including putative null alleles, transform the fates of certain L1 and L2 cells to those of L2 and L3 cells, respectively. Certain alleles have shown that the L1 and L2 effects of lin-14 are independently mutable: lin-14(a) alleles selectively affect the fates of L1 cells and lin-14(b) alleles affect only cells after the L1. It was proposed that these independently mutable lin-14 functions might selectively affect the level of a single gene product at particular stages (Ambros and Horvitz 1987). According to this view, three levels of lin-14 gene activity would confer three different lineage-specific fates on cells through the L1−L3 stages. Alternatively, lin-14 (a) and lin-14 (b) mutations might selectively affect the function of distinct gene products that are each separately down-regulated (Ambros and Horvitz 1987). Several different lin-14 transcripts have been identified from cDNA clones, and these could encode proteins that differ at their amino termini (Wightman et al. 1991). It is not yet known whether these transcripts are subject to tissue-specific or stage-specific regulation. It will be interesting to see if lin-14 does indeed produce multiple protein products with distinct expression patterns or if different levels of a single lin-14 gene product can direct normal development.
B. lin-4 Down-regulates LIN-14 Protein
Genetic and molecular experiments indicate that lin-4 is a negative regulator of lin-14 which has a critical role in lin-14 down-regulation (Ambros 1989). A lin-4(0) mutation causes a retarded phenotype essentially identical to that of lin-14(gf) mutations (Chalfie et al. 1981; Ambros and Horvitz 1987), and epistasis experiments indicate that these lin-4 defects depend on lin-14 function (Ambros 1989). Furthermore, lin-4 activity is required for the temporal decrease in LIN-14 protein level; in lin-4 mutant animals, LIN-14 protein remains abnormally high later in development (Arasu et al. 1991). Since lin-14 messenger RNA is not noticeably affected by lin-4 mutations (Wightman et al. 1993), these findings suggest a posttranscriptional role for lin-4 in lin-14 down-regulation. lin-14(gf) alleles contain deletions of sequences from the 3′-untranslated region (3′UTR) of the lin-14 transcripts. This suggests that the 3′UTR of lin-14 RNA contains a negative regulatory element that affects lin-14 activity posttranscriptionally, and through which the lin-4 gene product might act in trans (Ruvkun et al. 1989; Wightman et al. 1991).
lin-4 has been cloned, and, surprisingly, it does not encode a protein (Lee et al. 1993). It encodes two small untranslated RNAs, and the more abundant of the two transcripts, lin-4 S, is expressed beginning in the late L1 stage (R. Feinbaum and V. Ambros, in prep.). Since this is the time that LIN-14 protein begins to decrease, lin-4 S may be directly responsible for reducing the level of LIN-14 protein. lin-4 S RNA is 22 nucleotides long and contains two blocks of sequences that are complementary to an element repeated seven times in the 3′UTR of lin-14 mRNA, suggesting that lin-4 might inhibit LIN-14 protein synthesis by a complementary base pairing with lin-14 mRNA (Lee et al. 1993; Wightman et al. 1993; see also Anderson and Kimble, this volume). Although there has not yet been a direct demonstration of a lin-4 − lin-14 RNA interaction, the strong evolutionary conservation of the complementarity between lin-4 S and lin-14 3′UTR sequences in other nematode species strongly suggests that base pairing is important. The precise mechanism by which lin-4 S RNA down-regulates LIN-14 protein is not yet clear.
The temporal down-regulation of LIN-14 protein synthesis by lin-4 begins in response to signals that initiate larval development. In the absence of food, newly hatched L1 larvae do not develop; postembryonic cell divisions do not occur (Wood et al. 1988), and LIN-14 levels remain high (Arasu et al. 1991). After feeding, starvation-arrested larvae initiate postembryonic development, and LIN-14 protein decreases. The mechanism of action of this “food signal” that initiates postembryonic development is unknown, but it must ultimately lead to activation of lin-4 transcription because lin-4 RNA is not detected until after feeding of L1 animals (R. Feinbaum and V. Ambros, in prep.). It will be of great interest to identify the temporal signals that coordinate the activation of lin-4 transcription with L1 development.
C. Stage-specific lin-14 – lin-28 Interactions
Mutations in lin-28 result in defects generally similar to those of lin-14(b) alleles (Ambros and Horvitz 1984, 1987), where the L1 stage is unaffected, but the L2 stage events are deleted (Fig. 2A). Interestingly, lin-28 mutants seem to have an abnormally low level of LIN-14 protein during the late L1, consistent with a role for lin-28 in maintaining an appropriate level of LIN-14 expression (Arasu et al. 1991). These molecular observations, together with the similarity of lin-14(b) and lin-28 phenotypes, suggest that the lin-28 gene product might interact with the lin-14 gene or gene product during the late L1 stage to control L2-specific fates. The lin-28 gene product is not likely to function simply as an activator of lin-14 gene expression, because the lin-14 and lin-28 null alleles do not result in equivalent phenotypes. In lin-28(0) animals, some hypodermal cells can express an adult-specific event at the L2 molt, whereas in lin-14(0) mutants, that event is never expressed before the L3 molt (Ambros and Horvitz 1984; Ambros 1989). Furthermore, in the absence of lin-14 , the stage of expression of certain events still depends on lin-28 activity (Ambros 1989). For example, lin-28 is sufficient to prevent expression of L3-specific programs (S3) in the L1 even when lin-14 is absent (Fig. 2). Thus, lin-28 must have lin-14 -independent functions.
The functional relationship between lin-28 and lin-14 changes between the L1 and L2. Specifically, either lin-14 or lin-28 is sufficient to inhibit expression of S3 fates in the L1; neither single mutant alone expresses precocious S3 fates in the L1, but the double mutant does. In contrast, both lin-14 and lin-28 are required to inhibit S3 programs in the L2; the single mutants express precocious S3 programs at that stage. These observations suggest that perhaps the level of lin-28 activity also changes between the L1 and L2 stages. The recently cloned lin-28 gene was found to be a member of a family of nucleic-acid-binding proteins that contain “cold-shock” domains (E. Moss et al. in prep). The molecular characterization of lin-28 will allow tests of whether the lin-28 gene product interacts physically with LIN-14 and also should allow the identification of potential regulatory targets of LIN-28. The fact that members of the cold-shock domain family of proteins can have both RNA- and DNA-binding activities (Wolffe 1994) leaves open the question of whether lin-28 regulates gene expression at the transcriptional and/or posttranscriptional level.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY)
Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997. Section III, Control of Larval Development by lin-14.