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First published online 8 April 2004
doi: 10.1242/dev.01098

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The C. elegans heterochronic gene lin-46 affects developmental timing at two larval stages and encodes a relative of the scaffolding protein gephyrin

¹ Fox Chase Cancer Center, Philadelphia, PA 19111, USA
² Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA
³ Department of Molecular Biology, UMDNJ, Stratford, NJ 08084, USA

View larger version (23K): [in a new window]   Fig. 1. Lineages of hypodermal cells in heterochronic mutants. Schematics of post-embryonic cell lineages for lateral (seam; Vn=V1-V4 and V6) and ventral (Pn=P3-P8) cells are shown for animals of each genotype indicated. Vulva precursor cells (VPC) are the posterior daughters of P5-P7. A horizontal line depicts cell division. To the left in each set of lineages are indicated the cell-type specific lineage pattern interpreted from the data: 1, L1-specific cell lineage pattern (etc.); A, adult. 2/3 and 4/A indicate that cells at the indicated stage may execute a mix of alternative fates (see text). Broken lines indicate cells not depicted. Three horizontal lines indicate fusion of seam cells and secretion of adult lateral alae. The anterior daughters in Vn (seam) lineages, indicated by short lines, appear to fuse with the main hypodermal syncytium. Wild-type and lin-28(lf) lineages are taken from previous studies (Sulston and Horvitz, 1977; Ambros and Horvitz, 1984).

View larger version (21K): [in a new window]   Fig. 5. Genetic and molecular models for LIN-46 activity in developmental timing. (A) Genetic models for the heterochronic pathway governing fate choices at two stages. (Left) Interactions governing the L2/L3 fate choice. The circled regulators x and y have not been identified and may in fact represent multiple factors. lin-14 is depicted in its role in the L2/L3 fate decision only (lin-14B activity). The position of daf-12 is based on previous studies (Antebi et al., 1998; Seggerson et al., 2002); its role does not appear to be analogous to that of lin-46. (Right) Interactions governing the L/A switch. The effect of lin-28 or lin-46 on let-7 expression or activity has not been determined, and may be indirect. The linear pathway from let-7 to adult fates is based on previous studies (Abrahante et al., 2003; Lin et al., 2003). (B) A molecular model for LIN-46 protein function. LIN-46 may scaffold a multiprotein assembly constituting a developmental timing complex that regulates the expression of downstream heterochronic genes and promotes L3- and adult-specific cell fates. Broken arrow indicates that the effects of the complex may be either positive or negative, and either direct or indirect. LIN-28 may act as a negative regulator of one or more components of that complex, probably at the level of gene expression.

View larger version (42K): [in a new window]   Fig. 3. Cloning of lin-46 and its homology to MoeA and gephyrin. (A) Regions of the genetic and physical maps of linkage group V of C. elegans depicting the location of lin-46. Two cosmids shown rescued a lin-46 mutant. One of these was subcloned to yield the minimal rescuing BglI-NgoMI restriction fragment. The gene structure was determined by RT-PCR and sequence analysis. (B) An alignment of the predicted amino acid sequences of LIN-46 of C. elegans and C. briggsae. Black, identical residues; gray, similar residues. Vertical lines indicate exon boundaries. Arrows indicate positions of lesions in the mutant alleles: ma164, ma174 and ve1. Dots and asterisks indicate residues partially and highly conserved among gephyrin/MoeA homologues, respectively. (C) Schematics of LIN-46 related proteins: gephyrin, mammals; Cinnamon, Drosophila; CNX1, Arabidopsis; MoeA and MogA, E. coli; MOC-1, MOC-2 and LIN-46, C. elegans. MoeA-like (E) and MogA-like (G) regions are indicated.

View larger version (63K): [in a new window]   Fig. 2. Retarded development of the lateral hypodermis in a lin-46 mutant. A DIC micrograph of an adult animal of the genotype lin-46(ma164) grown at 20°C showing the same field in two planes of focus. Left, a pair of recently divided lateral hypodermal seam cells. Right, the gap in adult lateral alae overlying these cells. These gaps initially overly an even number of seam nuclei, but later, the anterior cell fuses with the main hypodermal syncytium.

View larger version (93K): [in a new window]   Fig. 4. Expression of lin-46:GFP in hypodermal cells during larval development. Fluorescence and DIC micrographs of animals bearing lin-46:GFP transgenes. Transgene arrays include a rol-6 marker that causes twists in the body. (A) L2 animal expressing lin-46:GFP transcriptional fusion showing fluorescent lateral hypodermal seam cells. (B) An L4-stage animal bearing the transcriptional fusion showing fluorescent seam cells apparently undergoing fusion. (C) L2 animals expressing a lin-46:GFP translational fusion. Fluorescence is seen in seam cells as well as two neurons (see text). The exposure time is three to eight times longer than for animals expressing transcriptional fusion. (D) Higher magnification showing nuclear and cytoplasmic localization of LIN-46:GFP in an L2 animal expressing the translational fusion. Pairs of seam cells often show similar fluorescence intensity. Scale bars: in A, 100 µm for A-C; in D, 10 µm.