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The zinc finger protein REF-2 functions with the Hox genes to inhibit cell fusion in the ventral epidermis of C. elegans

Department of Biochemistry and Biophysics, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0448, USA

View larger version (26K): [in a new window]   Fig. 1. The Pn.p cell fusion pattern is regulated by the Hox genes lin-39 and mab-5. (A) Ventral views of early-L1 (top) and mid-L1 (bottom) larvae. Six pairs of epidermal P blast cells initially lie along the ventral surface (top). During L1, the P cells migrate around each other so that a row of 12 P cells lines the ventral surface (bottom). Shortly after they migrate into the ventral cord, the P cells divide. The anterior (Pn.a) daughters become neuroblasts, while the posterior (Pn.p) daughters remain epidermal. Anterior is towards the left in this and all figures. (B) Lateral view of a late L1 larva with the 12 ventrally located Pn.p cells shown. Some of the Pn.p cells fuse with hyp7, which is located more dorsally along most of the mid-body region. (C,D) Pn.p cell fusion in wild-type hermaphrodite (C) and male (D). White circles indicate Pn.p cells that remain unfused, short horizontal lines indicate cells that fuse with the hyp7 syncytium. Hox gene expression domains are similar in both sexes, as indicated in hatched regions. The tables underneath show how Hox gene expression information is interpreted in the cell fusion decision. (B-D) Reproduced, with permission, from Alper and Kenyon (Alper and Kenyon, 2001).

View larger version (16K): [in a new window]   Fig. 2. (A) ref-2(mu218) was mapped to the black regions in the top two panels, as described in Materials and Methods. Ultimately, an 8 kb PCR product was identified that induced the mutant phenotype in the progeny of injected worms (bottom panel). Only a single open reading frame is predicted on this DNA. The verified cDNA structure is indicated with exons depicted as boxes. Gray boxes indicate the region where the five zinc fingers are encoded. The 3' UTR is indicated by hatching. The GA transition present in mu218 DNA is also depicted. Scale is indicated in the right-hand corner of each panel. (B) Anti-rescue was achieved by injecting DNA containing the mu218 GA transition (middle) but not by injecting wild-type DNA (top), or DNA containing both the GA transition and the TGAT insertion (which introduces stop codon and a frameshift prior to the first zinc finger). Anti-rescue indicates that P7.p and P8.p remained unfused in males.

View larger version (64K): [in a new window]   Fig. 3. P7.p and P8.p remain unfused in ref-2(mu218) males. (A,B) Unfused Pn.p cells are inferred from the presence of ventral protrusions (indicated by white arrows) in a lin-12(n137); him-5(e1467) worm. lin-12(n137) males (A) contain a gap in the ventral protrusion pattern that corresponds to the region where P7.p and P8.p have fused with hyp7. No gap in the protrusion pattern is present in lin-12(n137); ref-2(mu218) males (B). (C,D) Unfused Pn.p cells visualized by immunostaining animals with the MH27 monoclonal antibody. P(3-6).p and P(9-11).p remain unfused in wild-type males (C), while P(3-11).p remain unfused in ref-2(mu218) mutant males (D).

View larger version (33K): [in a new window]   Fig. 4. The ref-2(mu218) mutation prevents LIN-39 and MAB-5 from canceling each other’s activities in males. The indicated mutants were stained with MH27 in early L2 to score Pn.p cell fusion in males. White circles indicate unfused Pn.p cells, short horizontal lines indicate Pn.p cells that have fused with hyp7. Hox gene expression patterns in each set of Hox mutant strains are indicated above each set of genotypes. Alleles used: lin-39(n1760) and mab-5(e2088), both null mutations, and mab-5(e1751), in which wild-type mab-5 is misexpressed in all Pn.p cells. All strains also harbored the him-5(e1490) mutation to generate males. At least 50 worms of each genotype were scored. All phenotypes listed are 100% penetrant except for: in mab-5(e1751); him-5(e1490) worms, P1.p fused with hyp7 in 1/52 worms and P3.p remained unfused in 1/52 worms, and in mab-5(e1751); him-5(e1490); ref-2(mu218) worms, P6.p fused with hyp7 in 1/51 worms.

View larger version (62K): [in a new window]   Fig. 5. ref-2 encodes a protein with five zinc-finger domains similar to the opa/Zic family of zinc-finger genes. (A) The region around the mu218 GA transition is depicted. The mutation lies 399 bp beyond the last base in the 3' UTR listed in B. (B) Sequence and inferred translation of ref-2 cDNA determined by sequencing of RT-PCR products. The five C2H2 zinc finger domains are underlined. The location of introns is indicated by black triangles. A putative polyadenylation signal is underlined twice. (C) Alignment of the zinc-finger region of REF-2 with the zinc finger region of the related proteins Xenopus Zic2 (Nakata et al., 1998), human ZIC1 (Yakota et al., 1996), mouse Zic3 (Aruga et al., 1996) and Drosophila Opa (Benedyk et al., 1994; Cimbora and Sakonju, 1995). Residues conserved in at least 50% of sequences are highlighted in black. Asterisks below the sequences indicate the conserved cysteines and histidines. Data analysis was carried out using Pileup and Boxshade (GCG Wisconsin Package version 10.1, Genetics Computer Group, Madison, WI).

View larger version (9K): [in a new window]   Fig. 6. Wild-type ref-2 is required for Pn.p cells to be generated and to remain unfused. ref-2 gene function was inhibited by RNAi as described in the Materials and Methods. A range of phenotypes was observed depending on the concentration of dsRNA injected. Weaker RNAi produced worms in which most of the Pn.p cells had fused (short horizontal lines), Stronger RNAi produced worms in which most of the Pn.p cells were absent from the ventral cord (A). White circles indicate unfused Pn.p cells. Pictured are data for hermaphrodites; similar phenotypes were also observed in males.

View larger version (37K): [in a new window]   Fig. 7. ref-2 is expressed in the P cell lineage in wild-type hermaphrodites. Staged population of L1 larvae were stained with an anti-REF-2 antiserum (red in A,D,G,J) and the MH27 monoclonal antibody (green in B,E,H,K) as described in the Materials and Methods. Merged images are shown in C,F,I,L. REF-2 is initially detected in the 12 P cells just prior to their migration into the ventral cord (A-C; ventral view) and remains on as the P cells migrate (D-F; ventral view). In this picture, P1 and P2 have migrated; the other P cells will migrate soon. REF-2 is then present in both daughters of all P cells following division (G-I; ventrolateral view). REF-2 then disappears in the Pn.a cells and their progeny. REF-2 also disappears in the Pn.p cells, although it does so at different rates. REF-2 remains on longest in the six unfused Pn.p cells P(3-8).p (J-L, lateral view). White arrowheads indicate P1/2, P3/4,..P9,10 in A, P1 and P2 (which have migrated) and P3/4, P5/6, P7/8, P9/10 in D, and P(3-8).p in J.

View larger version (11K): [in a new window]   Fig. 8. ref-2 is expressed dynamically in the P cell lineage. The figure summarizes the REF-2 staining pattern in the P cell lineages during L1 in wild-type hermaphrodites. Black squares indicate REF-2 present, white squares indicate REF-2 absent, squares that are half filled indicate that weak REF-2 staining can be detected at this stage in some worms.

View larger version (45K): [in a new window]   Fig. 9. ref-2 expression correlates with the pattern of Pn.p cell fusion in wild-type and mutant worms. Staged populations of the indicated mutants were stained with an anti-REF-2 antiserum. The Pn.p cell fusion pattern is indicated by white circles (unfused cells) and short horizontal lines (fused cells) next to the genotype. Below each cell is the anti-REF-2 staining pattern in the Pn.p cells starting just after the Pn.p cells are formed. The first two time points (first two rows) are before Pn.p cell fusion, the last four are after the time of Pn.p cell fusion. Black squares indicate that REF-2 was detected at that time in those cells, white squares indicate no REF-2 was detected and half filled squares indicate weak expression was sometimes detected.

View larger version (20K): [in a new window]   Fig. 10. Two stages in ref-2-dependent patterning of Pn.p cell fusion. (A) A model for how REF-2 prevents Pn.p cell fusion. REF-2 and a Hox protein (LIN-39 or MAB-5) act by cooperatively inhibiting a common set of fusion gene targets. Alternatively, the two transcription factors could together activate a gene(s) required to prevent cell fusion. (B) A model for how ref-2 expression might be regulated. In Pn.p cells in males, either Hox protein alone (LIN-39 or MAB-5) can activate ref-2 transcription and thus can keep the concentration of REF-2 high in the Pn.p cells when the cell fusion decision is made. However, when both Hox proteins are present, ref-2 expression decreases in the Pn.p cells and those cells fuse with hyp7. The mu218 mutation allows ref-2 to be expressed even in cells containing both LIN-39 and MAB-5 (mu218 could affect the ability of Hox proteins to bind ref-2 regulatory sites).