Structure and function analysis of LIN-14, a temporal regulator of postembryonic developmental events in Caenorhabditis elegans

Abstract

During postembryonic development of Caenorhabditis elegans, the heterochronic gene lin-14 controls the timing of developmental events in diverse cell types. Three alternative lin-14 transcripts are predicted to encode isoforms of a novel nuclear protein that differ in their amino-terminal domains. In this paper, we report that the alternative amino-terminal domains of LIN-14 are dispensable and that a carboxy-terminal region within exons 9 to 13 is necessary and sufficient for in vivo LIN-14 function. A transgene capable of expressing only one of the three alternative lin-14 gene products rescues a lin-14 null mutation and is developmentally regulated by lin-4. This shows that the deployment of alternative lin-14 gene products is not critical for the ability of LIN-14 to regulate downstream genes in diverse cell types or for the in vivo regulation of LIN-14 level by lin-4. The carboxy-terminal region of LIN-14 contains an unusual expanded nuclear localization domain which is essential for LIN-14 function. These results support the view that LIN-14 controls developmental timing in C. elegans by regulating gene expression in the nucleus.

FIG. 1

Genomic organization of wild-type lin-14 and lin-14 deletion constructs. (A) lin-14 intron-exon structure of the three identified lin-14 transcripts, lin-14B1, lin-14B2, and lin-14A (adapted from reference 28). Restriction enzyme map and intron-exon spacing are drawn to scale. Open box in exon 4 indicates that the 5′ end of the exon 4 open reading frame is undetermined. A, AgeI; B, BglII; E, EcoRI; K, KpnI; S, SalI. (B and C) LIN-14 deletion-GFP fusion constructs p14GFP and p14B2GFP. A single line represents a region that is deleted. E∗, EcoRI site that was eliminated (see Materials and Methods). (D) Truncated lin-14 constructs expressed from the col-10 promoter. col-10 promoter sequences and 3′UTR sequence of lin-14 are not shown. Open boxes indicate introns and filled boxes indicate exons. GFP sequence is represented by the hatched box (artificial introns in GFP are not shown). Amino acids are numbered according to Wightman et al. (28). D10 contains the lin-14(n179ts) mutation (R303G in exon 9; B. Reinhart and G. Ruvkun, personal communication; see Materials and Methods).

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. 1). High levels of green LIN-14::GFP fluorescence is evident in hypodermal cells, muscle cells, intestinal cells, and neurons. Arrowheads point to nuclei that show punctuate LIN-14::GFP fluorescence typical of this construct. The yellow fluorescence seen in these images is due to the autofluorescence from the intestine. (B) LIN-14::GFP expression in maEx166 animals becomes virtually undetectable in hypodermal cells (hyp) by the L3 stage. Yellow signal is (non-GFP) intestinal autofluorescence. (C) In maEx166 animals, LIN-14::GFP expression in the head neurons remains detectable in some L3 animals. (D) lin-4(e912); maEx166 L1 larvae display fluorescence levels approximately equal to that of lin-4(+); maEx166 L1 larvae. (E) At the L3 stage, high levels of LIN-14::GFP expression persists in hypodermal cells and in intestinal cells of lin4(e912); maEx166 L3 larvae. (F) Expression of LIN-14::GFP in VPC (Pn.p) cells is easily detectable in this lin-4(e912); maEx166 L2 larva [but is undetectable in similarly staged lin-4(+); maEx166 L2 larvae; not shown]. Animals in B, E, and F are all oriented anterior down and ventral side to the right. Bar, 3 μm.

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 exon in C. elegans LIN-14 is labeled. Black lines highlight the potential consensus sequences for nuclear localization activity (15). The white line between the two sequences within exon 11 indicates the putative amphipathic helix motif (28). Also shown is the n179 point mutation (R303G) (B. Reinhart and G. Ruvkun, personal communication). Note that sequences shown here are equivalent to the predicted lin-14B1 product (see Fig. 1A) and the very amino-terminal end of the C. vulgaris LIN-14 sequence is undetermined. C. vulgaris sequence was determined from cDNA.

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 (at 15°C) (H), and LIN-14D10::GFP (at 25°C) (I). Animals were at developmental stages ranging from L1 to L3. (E and F) Unlocalized LIN-14D7::GFP and LIN-14D8::GFP appear as a uniform green fluorescence, while in all other panels except A; the green fluorescent spots are nuclei in which LIN-14::GFP is localized. Nucleoli are relatively free of LIN-14::GFP staining and appear as darker spots within the nuclei. The nuclear localization patterns of LIN-14D1::GFP and LIN-14D2::GFP (data not shown) are similar to that of LIN-14D4::GFP. (A) Lateral hypodermal seam cells (arrow) are filled with unlocalized LIN-14D3::GFP and show a higher level of GFP fluorescence than surrounding hypodermal cells, and the inset shows the unidentified GFP-containing inclusions that are frequently evident in animals expressing LIN-14D3::GFP. We have not determined whether the structures are extracellular or intracellular. The image was captured with relatively short exposures (1/30 or 1/60 s) so the actual level of GFP fluorescence is much higher than in the rest of panels, which were taken at exposures ranging from 1/4 to 2 s. (J) Quantitative assay of the nuclear localization efficiency of truncated LIN-14::GFP proteins. In the diagram on the left, black regions in exon boxes represent the amino acid sequences that are relatively well conserved among LIN-14 proteins from different nematode species (see Fig. 3). GFP sequences are not shown, and the intron spaces in D3 are not drawn in scale. N/C, nuclear-to-cytoplasmic ratio of GFP fluorescence plotted in a log scale (see Materials and Methods). LIN-28::GFP is a cytoplasmically localized protein (4) used here as a control. Bar, 5 μm.

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 mitosis, and the arrowheads in this and subsequent frames highlight the mitotic chromosomes. Larger arrows point to new sister nuclei. LIN-14D4::GFP is completely nuclear localized in surrounding interphase hypodermal cells. Bar, 3 μm.