A LIN-14::GFP fusion construct that can fully rescue a lin-14 null mutation. lin-14
sequences span approximately 20 kb of genomic DNA (28
) (Fig. A). To analyze the functional organization of LIN-14 protein, a modified lin-14
genomic construct, p14GFP, was constructed (Fig. B). p14GFP encompasses lin-14
genomic DNA from 5.2 kb upstream of exon 1 to about 500 bp downstream of the polyadenylation signal, with a deletion of approximately 7 kb of intron sequence between exon 3 and exon 4 (Fig. B; see Materials and Methods). p14GFP also contains a GFP sequence inserted in frame at the LIN-14 carboxy terminus (see Materials and Methods) to enable the detection of LIN-14::GFP fusion protein in transgenic worms.
p14GFP was transformed into lin-14(ma135)
animals, a lin-14
genetic null (18
; V. Ambros, unpublished data) and stable transgenic lines were established. The anatomical pattern of expression of p14GFP from the transgenic extrachromosomal array maEx167
is consistent with that of the endogenous LIN-14 protein as previously determined by LIN-14 antibody staining (23
) (see below). LIN-14::GFP was nuclear localized, as is the case for endogenous LIN-14 (23
) (see below).
As shown in Tables and , lin-14(ma135); maEx167 animals were fully rescued for all defects scored, including precocious L1 cell lineage patterns in the V lineage, precocious seam cell differentiation, precocious vulva development, and precocious initiation of dauer larval development (Tables and ).
TABLE 1 Rescue of lin-14(lf) phenotypes by p14GFP and p14B2GFPtransgenes
TABLE 2 Rescue of precocious dauer formation by p14GFP and p14B2GFP in lin-14(n179ts) and lin-14(ma135)mutants Alternative exons 2 and 4 are not required for lin-14 activity.
p14GFP should be capable of making all three lin-14
, and -B2
) since it contains the alternative exons 2 (which is B1
specific) and 4 (which is A
specific). To test whether lin-14
products containing those alternative exons are required for lin-14
function, we examined the rescuing activity of a construct (p14B2GFP) that is missing exons 2 and 4. p14B2GFP was generated by the deletion from p14GFP of an approximately 1.0-kb DNA sequence around (and including) exon 2 and the deletion of a 500-bp DNA sequence containing exon 4 (Fig. C). While it is impossible for p14B2GFP to make either lin-14A
mRNA, GeneFinder (9
) predicts that this construct should produce the lin-14B2
transcript (data not shown).
The overall expression pattern of GFP from maEx168
is similar to that from maEx166
(see below) and maEx167
, indicating that no tissue-specific promoter sequences were affected by the p14B2GFP deletions (data not shown). maEx168
, an extrachromosomal array carrying p14B2GFP, was tested for its ability to rescue lin-14(n179ts). n179
is a temperature-sensitive (ts) allele of lin-14
, and at 25°C, lin-14(n179ts)
animals exhibit the lin-14(lf)
), though the phenotype is somewhat less severe than that of lin-14(ma135)
(see Table ). As summarized in Table , maEx168
fully rescues lin-14(n179ts)
for all the phenotypes tested.
The temperature-sensitive lin-14(n179ts) mutation does not completely eliminate lin-14 activity at 25°C. To exclude the possibility that p14B2GFP might rescue lin-14(n179ts) by boosting the weak residual activity of the temperature-sensitive LIN-14 protein, we crossed maEx168 into animals carrying the non-temperature-sensitive allele lin-14(ma135) and found efficient rescue of lin-14(ma135) precocious defects (Table ). These results provide strong evidence that neither exon 2 or exon 4 is required for lin-14 activity and that a single predicted product of lin-14 is sufficient for LIN-14 function in different cell types.
lin-4-dependent developmental down regulation of LIN-14::GFP expression from transgenes.
The successive execution of larval stage-specific developmental programs requires the temporal down regulation of lin-14 activity between the L1 and L2 stages. We found that the effective level of LIN-14::GFP expressed from maEx167 is within the normal range (in terms of lin-14 activity) and appears to be down regulated satisfactorily to achieve the normal sequence of larval developmental events. The phenotype of maEx167 animals showed no significant evidence of LIN-14 overexpression, as seam cells made adult cuticle at the L4 molt and the timing of vulva development was normal (Table ).
The temporal down regulation of lin-14
activity is executed at the posttranscriptional level. This down regulation requires the product of the heterochronic gene lin-4
, which encodes a small 22-nucleotide RNA with sequence complementarity to seven elements in lin-14
). To confirm that LIN-14::GFP expression from transgenic p14GFP is down regulated by lin-4
, LIN-14::GFP expression was monitored in wild-type and lin-4(e912)
, a transgenic array similar to maEx167
, was generated by coinjecting p14GFP along with pRF4 (rol-6(su1006)
). In wild-type maEx166
animals, the temporal profile of LIN-14::GFP expression faithfully reflects the results previously obtained by LIN-14 antibody staining of the wild type (23
animals displayed the highest LIN-14::GFP expression in the nuclei of late embryos just before hatching and of newly hatched L1 animals (Fig. A). This high level of LIN-14::GFP expression starts to decrease in hypodermal and intestinal nuclei and in the nuclei of ventral nerve neurons during the L1 stage and becomes undetectable in the L2 stage (Fig. B). The only exception is a subset of neurons in the head in which LIN-14::GFP remains detectable as late as the L3 or L4 stage (Fig. C). The expression of LIN-14B2::GFP from maEx168
showed a temporal down regulation essentially indistinguishable from that of LIN-14::GFP expressed from maEx166
(data not shown).
FIG. 2 lin-4-dependent down regulation of LIN-14::GFP from maEx166. (A) Newly hatched transgenic L1 larva carrying maEx166, which was generated by injection of p14GFP (Fig. ). High levels of green LIN-14::GFP fluorescence is evident in hypodermal (more ...)
To test whether the down regulation of LIN-14::GFP from maEx166 or LIN-14B2::GFP from maEx168 is lin-4 dependent, we crossed maEx166 and maEx168 into a lin-4(e912) background. In lin-4(e912); maEx166 animals, LIN-14::GFP expression was poorly down regulated compared to the wild type and was easily detectable in hypodermal and intestinal nuclei in animals at stages as late as the L3 or L4 (Fig. D to F). This indicates that down regulation of LIN-14::GFP from maEx166 requires lin-4 activity, as is the case for LIN-14. Similarly, down regulation of LIN-14B2 was not evident in lin-14(e912); maEx168 animals. These results indicate that the 7 kb of intron sequences deleted in p14GFP and the additional sequences deleted from around exons 2 and 4 in p14B2GFP (Fig. ) are dispensable for LIN-14 expression and temporal regulation by lin-4.
Interestingly, although wild-type VPCs display no detectable levels of endogenous LIN-14 (23
) or LIN-14::GFP (data not shown), LIN-14::GFP was easily detected in VPCs during the late L1 to L2 stage in lin-4(e912)
animals (Fig. F). The expression of LIN-14 in lin-4(e912)
VPCs is consistent with the retarded vulva phenotype of lin-14(e912)
animals and the critical role of LIN-14 down regulation in regulating the competence and cell cycle progression of VPCs (10
Exons 9 to 13 of lin-14 are sufficient for lin-14 activity in lateral hypodermal cells.
To further delineate the parts of LIN-14 protein that are necessary and sufficient for its in vivo function, a series of deletions of the LIN-14::GFP fusion protein were constructed (Fig. D). In these experiments, LIN-14 deletion constructs were expressed from a simplified expression vector under the control of the C. elegans col-10
is specifically expressed in hypodermal cells (19
), and thus the rescuing activity of LIN-14 deletion constructs was scored in the hypodermis. The assays were performed in a lin-14(n179ts)
background because the temperature-sensitive phenotype of lin-14(n179ts)
allows efficient transformation at permissive temperature (15°C) and a convenient assay for rescue simply by transferring the transgenic animals to nonpermissive temperature (25°C). It should be noted that although the injection conditions were similar for each construct (see Materials and Methods), the expression levels of these truncated LIN-14 proteins in general were greater than the LIN-14::GFP levels observed from maEx167
(see Fig. and ). In particular, the expression level of LIN-14D3::GFP was very high (see Fig. A).
FIG. 4 Nuclear localization efficiency of various truncated LIN-14::GFP proteins. (A to I) Hypodermal cells expressing LIN-14D3::GFP (A) LIN-14D4::GFP (B), LIN-14D5::GFP (C), LIN-14D6::GFP (D), LIN-14D7::GFP (E), LIN-14D8::GFP (F), LIN-14D9::GFP (G), LIN-14D10::GFP (more ...)
Surprisingly, although lin-14 contains a total of 13 exons, we found that a deletion construct containing only exons 9 to 13 (Fig. D, construct D5) rescued lin-14(n179ts) at 25°C (Table and data not shown). Similarly, deletion construct D9 (containing exons 8 through 12) rescued lin-14(n179ts), though not as strongly as D5 (Table ). This suggests that sequences sufficient for lin-14 activity are contained within exons 9 to 12, although some of exon 13 may also contribute. Sequences in exon 9 appear to be critical for lin-14 activity, as construct D6 (containing exons 10 to 13; see Fig. D) showed no rescuing activity (Table ). Construct D8 (containing exons 9 and 10 and part of exon 11; see Fig. D) also showed no rescuing activity, indicating that sequences in the exon 11-to-12 interval are essential. These findings indicate that sequences contained within a carboxy-terminal region of LIN-14 encoded by exons 9 to 13 are both necessary and sufficient for lin-14 activity, as assayed by rescue of lin-14(n179ts) defects in the hypodermis. Exons 9 to 12 corresponds to a part of LIN-14 where the amino acid sequence is well conserved among the nematode species C. elegans, Caenorhabditis vulgaris (Fig. ), and Caenorhabditis briggsae (B. Reinhart and G. Ruvkun, personal communication), consistent with the functional activity of this region of the protein.
FIG. 3 Amino acid sequence alignment between LIN-14 proteins from C. elegans (ele) and C. vulgaris (vul). Identical amino acids are labeled in black boxes, and dashes indicates gaps generated by the aligning program GeneInspector. The start position of each (more ...)
To exclude the possibility that the rescuing activity of the above constructs depends on endogenous residual activity of LIN-14(n179ts) protein, for example, by synergizing with or stabilizing the temperature-sensitive LIN-14 protein, we tested the rescuing activity of LIN-14D5::GFP in lin-14(ma135) animals, which carry a non-temperature-sensitive null allele of lin-14. In lin-14(ma135) animals carrying a transgenic array of construct D5, 60.4% of seam cells are rescued from making precocious adult cuticle at the L3 stage. These data are comparable to the rescuing activity of LIN-14D5::GFP protein in lin-14(n179ts) (Table , ~73%), demonstrating that the product of exons 9 to 13 alone supplies lin-14 activity.
Truncated LIN-14::GFP fusion proteins, particularly D4 to D10, are generally not well down regulated during larval development. Unlike animals transformed with full-length LIN-14::GFP constructs (see above), animals carrying truncated constructs often exhibit bright fluorescence after the L1 stage (see Fig. ). Truncated rescuing constructs can also cause retarded phenotypes of varying strengths, although we have not quantitatively compared the truncated constructs to the full-length constructs with respect to retarded phenotypes (data not shown).
FIG. 5 LIN-14D4::GFP is efficiently localized to nuclear material during mitosis. A dividing cell in a transgenic lin-14(n179ts) at the late L2 stage expressing LIN-14D4::GFP at 20°C. Small arrow in 0 min image points to a hypodermal cell undergoing (more ...) Partial dominant negative activity of truncated LIN-14 products.
lin-14(n179ts) at permissive temperature (15°C) provides a sensitized genetic background for testing the potential antimorphic (“dominant negative”) activity of the truncated LIN-14 proteins produced by these deletion constructs. We have found that at 15°C, approximately 13% of seam cells in n179 animals express precocious adult cuticle (Table ), and this weak precocious phenotype indicates that at 15°C, the level of lin-14 activity in lin-14(n179ts) animals might be slightly below the threshold required for wild-type seam cell development. Such a leaky precocious phenotype of lin-14(n179ts) would be enhanced in the presence of a truncated LIN-14 protein which is able to interfere with endogenous lin-14 function.
lin-14 deletion constructs were tested for their ability to either rescue or enhance the leaky precocious phenotype of lin-14(n179ts) animals at 15°C. As shown in Table , rescuing constructs clearly prevent the formation of precocious adult cuticle in lin-14(n179ts) animals at 15°C, but one of the nonrescuing constructs, D6 (which contains exons 10 to 13), enhances the leaky precocious phenotype. In lin-14(n179ts) animals carrying the D6 transgenic array, more seam cells express precocious adult cuticle at 15°C than in lin-14(n179ts) animals (Table ). This antimorphic, or partial dominant negative, activity of LIN-14D6::GFP protein requires exon 10, since a further truncated construct (D7) containing only exons 11 to 13 does not modify the lin-14(n179ts) phenotype. The antimorphic activity of LIN-14D6::GFP seems to require the sensitized lin-14 hypomorphic genetic background provided by lin-14(n179ts), since no such activity was observed in a wild-type background.
LIN-14D3::GFP, which contains LIN-14 sequences from exons 5, 6, and 7 and to the beginning of exon 8 (Fig. D), shows very weak antimorphic activity in lin-14(n179ts) animals at 15°C (Table ). The relatively low strength of the LIN-14D3::GFP negative activity, coupled with the extraordinarily high level of expression of this construct relative to that of D6, casts doubt on the specificity of the D3 phenotype.
LIN-14 has an extended nuclear localization domain.
To characterize sequences required for the nuclear localization of LIN-14, we examined the nuclear localization of truncated LIN-14::GFP proteins. The ratio of nuclear GFP fluorescence to cytoplasmic GFP fluorescence was measured in transgenic worms carrying various LIN-14::GFP constructs (see Materials and Methods). Exons 1 to 7 do not seem to contain any essential nuclear localization signals (NLS), since LIN-14D4::GFP which contains exons 8 to 13 is efficiently nuclear localized (Fig. B and J). Further NLS sequences seem to be contained between the end of exon 8 and the beginning of 12, since LIN-14D9::GFP is also nuclear localized (Fig. G and J). LIN-14D5::GFP, which contains exons 9 to 13, is still nuclear localized, but its localization efficiency is less than that of LIN-14D::GFP (Fig. C and J), suggesting that nuclear localization of LIN-14 is influenced by sequences in exon 8. Further deletion of sequences from either the N terminus or C terminus diminishes the nuclear localization of LIN-14 significantly (Fig. D, E, F, and J).
These results suggest that LIN-14 nuclear localization requires sequences at approximately the exon 8 and 9 border and also requires sequences in the exon 11 and 12 region. Thus, the LIN-14 NLS is either bipartite or extends over a region of LIN-14 from exon 8 to exon 12. Basic Arg-Lys clusters similar to a typical NLS consensus sequence are found at both ends of this region (Fig. ), but the above results show that neither of these regions alone is sufficient to bring LIN-14 to the nucleus. Interestingly, LIN-14D10::GFP, which contains exons 9 to 13 with the n179 point mutation (B. Reinhart and G. Ruvkun, personal communication), is localized to nuclei (Fig. ), despite have in no rescuing activity or antimorphic activity (Table ). This suggests that the n179 mutation impairs a component of LIN-14 function other than nuclear localization. LIN-14 sequences required for nuclear localization could function by interacting directly with the nuclear localization machinery, or indirectly, via a nuclear-transported partner.
The behavior of LIN-14::GFP fusion proteins during cell division suggests that LIN-14 nuclear localization is rapid and efficient. For example, after premitotic nuclear envelope breakdown, the truncated LIN-14 fusion protein LIN-14D4::GFP appears uniformly distributed in the cytoplasm and then becomes reconcentrated in the daughter nuclear material during a brief period shortly after metaphase (Fig. ). This observation suggests that the process of LIN-14 nuclear localization is very efficient and may involve interaction with some component of the mitotic apparatus. A small amount of the LIN-14D4::GFP fluorescence during metaphase appears to be associated with chromosomes, suggesting a possible chromatin binding activity of the truncated protein (Fig. ). However, we did not observe a similar mitotic chromatin association of the full-length LIN-14::GFP and LIN-14B2::GFP proteins (data not shown). The relatively lower level of overall expression of LIN-14::GFP and LIN-14B2::GFP compared to that of the truncated LIN-14D4::GFP protein could account for the difficulty in detecting chromosome-associated fluorescence for the full-length fusion proteins. However, it is noteworthy that LIN-14::GFP and LIN-14B2::GFP fluorescence was not apparent anywhere within dividing hypodermal cells, yet was easily detectable before cell division and in daughter cell nuclei (data not shown). This suggests that full-length LIN-14 may be preferentially decreased in level in association with mitosis, while in contrast, the truncated LIN-14 proteins may be relatively stable during mitosis.