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Roles of the Homothorax/Meis/Prep homolog UNC-62 and the Exd/Pbx homologs CEH-20 and CEH-40 in C. elegans embryogenesis

Kimberly Van Auken^1,*, Daniel Weaver^1,, Barbara Robertson¹, Meera Sundaram², Tassa Saldi¹, Lois Edgar¹, Ulrich Elling^1,, Monica Lee¹, Queta Boese^1, and William B. Wood^1,¶

¹ Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA
² Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6145, USA
^* Present address: Department of Pediatrics, UCHSC, 4200 E. 9th Avenue, Denver, CO 80262, USA
Present address: Array BioPharma, 3200 Walnut St., Boulder, CO 80301, USA
Present address: EMBL Heidelberg, Meyerhofstrasse 1, Heidelberg D-69117, Germany
Present address: Dharmacon Research, 1376 Miners Drive, Lafayette, CO 80026, USA

View larger version (8K): [in a new window]   Fig. 6. Physical map of deletion and inversion breakpoints in the vicinity of the unc-62 promoter, showing numbered regions predicted to include possible regulatory elements (see Discussion). Exon sizes are not to scale.

View larger version (27K): [in a new window]   Fig. 1. unc-62 molecular cloning, transcript structure and sequence comparisons. (A) Genetic and physical maps of the unc-62 region on LGV. Genetic mapping placed unc-62 on the left arm of LGV, between two strain-specific polymorphisms stP3 and RW#L63 and in the region uncovered by deficiency sDf27 but not sDf50. Cosmids mapped to this region by the C. elegans genome consortium are shown below. unc-62 putative regulatory sequences and coding sequence reside on cosmids T08H10 and T28F12, respectively; both the regulatory and coding sequences are included in the fosmid clone H06N16. (B) Structure of unc-62 transcripts and locations of mutant lesions. unc-62 produces four alternatively spliced transcripts that differ in the choice of the first exon (1a or 1b) and the seventh exon, which is the first exon of the homeobox region (7a or 7b). The HM domain is encoded by exons 2, 3, 4 and 5, while the TALE homeodomain is encoded by exons 7a or 7b, 8 and 9. Locations of the point mutations t2012 and e644, the deletions s472 and ct344, and the left inversion breakpoint of e917 are indicated (see Fig. 6 for a more precise map of the rearrangements). (C,D) Sequence similarities of the conserved UNC-62, Homothorax, and murine Meis1 HM and homeodomains, respectively. Dashes indicate identical residues.

View larger version (48K): [in a new window]   Fig. 2. (A) Blots of RNA from early (pregastrulation) embryos (EE), mixed-stage embryos (ME), adult soma (AS) and adult total (AT) hybridized to four transcript-specific probes. EE: >90% pregastrulation embryos (Schauer and Wood, 1990). ME: mixed-stage embryos isolated by hypochlorite treatment of him-8(e1489) hermaphrodites. AS: glp-4(bn2ts) young adult hermaphrodites that had been shifted to non-permissive temperature (25°C) at hatching; these animals lack almost all germline cells (Beanan and Strome, 1992). AT: fem-2(b245ts) young adult hermaphrodites that had been shifted to non-permissive temperature (25°C) for 36 hours after hatching (the temperature-sensitive period), then returned to permissive temperature; these animals produce no sperm and therefore contain oocytes but no fertilized embryos (Kimble et al., 1984). Photographs of 18S rRNA UV absorption bands on the blots before probing are shown as loading controls (lower panels). The fourth (AT) lane of each gel is clearly overloaded relative to the other three. In preliminary experiments using equal specific activities for the 1a and 1b probes, the 1b bands were fainter than the 1a bands. In the experiment shown, a 1b probe at about twice the specific activity of the 1a probe was used in order to better compare band intensities among the four RNA preparations. (B) Results of real-time quantitative PCR experiments to compare the relative ratios of each transcript with total unc-62 mRNAs in the same RNA populations as analyzed in A. Figures in the table represent the ratio obtained for each primer pair relative to the ratio obtained for the EE sample, which was arbitrarily assigned a value of 1.0. The ranges shown for each ratio were calculated from the results for each triplicate set according to User Bulletin #2: ABI Prism 7700 Sequence Detection System (2001). This experiment was carried out three times with similar results; ratios and ranges shown in the figure are from one experiment.

View larger version (141K): [in a new window]   Fig. 3. Late embryonic and larval arrest phenotypes resulting from five unc-62 alleles. Animals in row A are homozygous mutant progeny of heterozygous unc-62/+ hermaphrodites; those in rows B-E are progeny of homozygous mutant hermaphrodites. Alleles are indicated in each panel. Each allele results in a range of phenotypes represented by the individuals shown.

View larger version (156K): [in a new window]   Fig. 4. Aberrant organization of early embryonic dorsal hypodermal and seam cell precursors and the resulting differentiated cells in unc-62 mutant embryos. Scale bars represent 10 µm. (A-D) Nomarski images of embryos (dorsal or ventral view, dorsal focal plane, anterior leftwards) that were lineaged to identify cells and photographed about 4 hours post-fertilization (22-25°C). Precursors of hypodermal cells derived from the C lineage and seam cells derived from V lineages are positioned as indicated. (A) Wild-type embryo. Note that dorsal C-derived hypodermal precursors are flanked by V-lineage cells. (Although this embryo is shown approximately one division earlier than the other embryos, the relative position of dorsal midline C hypodermal cells and lateral V precursors is already established at this time, and does not change.) (B) s472 mutant embryo. Some of the C hypodermal precursors lie abnormally ventral to this focal plane, and are not visible. (C) t2012 mutant embryo. (D) ct344 mutant embryo. Embryos in B-D are about one division later than the embryo in A. Relative positions of the C- and V-derived hypodermal cells are aberrant in the three mutants. (E-N) Nomarski and fluorescent images (lateral views, surface focal plane, anterior leftwards) of embryos and larvae expressing JAM-1::GFP, a marker for hypodermal cell junctions. (E,F) Wild-type newly hatched L1, showing the normal expression of JAM-1 at the borders of the row of seam cells. The dorsal and ventral hypodermal cells have fused into syncytia, showing no cell boundaries. (G,H) s472 newly hatched L1. There are few outlined cells, which do not show a contiguous seam cell pattern. (I,J) e644 L1, with a more normal pattern but some dorsal hypodermal cells that have not fused. (K,L) t2012 newly hatched L1, with disconnected seam cells. (M,N) ct344 late embryo, with few, abnormally large seam cells.

View larger version (103K): [in a new window]   Fig. 5. Examples of aberrant vulval development in ku234 mutant L4 hermaphrodites. (A) Wild type. (B) ku234: descendants of P7.p (arrow) have not joined in the main invagination. About 50% of ku234 exhibit this phenotype (non-Vul-abnormal), while the rest have apparently normal vulvae. (C) ku234/s472: descendents of P7.p (arrow) appear non-vulval. About a third of the non-Dpy segregants from ku234 dpy-11/unc-62(s472) unc-46 exhibited this semi-vulvaless phenotype, which is only slightly more severe that the non-Vul-abnormal phenotype in B. The remaining non-Dpy animals were either non-Vul-abnormal or normal. (D) ku234/sDf27: posterior Pn.p cells appear to be generating vulval-like descendants near the tail (arrow). This phenotype was seen only occasionally. As in C, about a third of the non-Dpy segregants of ku234 dpy-11/sDf27 unc-46 exhibited the semi-vulvaless phenotype, while the rest were non-Vul-abnormal or normal.