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First published online 17 October 2007
doi: 10.1242/dev.002741

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Co-regulation by Notch and Fos is required for cell fate specification of intermediate precursors during C. elegans uterine development

¹ Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.
² Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
³ The Honors College, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA.

View larger version (61K): [in this window] [in a new window]   Fig. 1. A minimal 1.3 kb enhancer in egl-13 URS drives expression specifically in the cell lineage. (A) The cells are singled out from a population of 12 VU intermediate precursors (six on one lateral side are shown, within 5 µm of the midline) surrounding the central AC, and undergo one round of cell division (d., dorsal; v., ventral). Eight descendants plus the AC form the utse syncytium; four descendants become uv1 and are more proximal to the vulva. (B) Medial uterine and vulval morphology. Base, basement membrane. (C) In a lateral plane to B, egl-13::GFP(tyIs4) initiates uterine expression only in the cells and persists in descendants. Fluorescent nuclei are bracketed. Asterisks mark fluorescent nuclei of the body wall. Scale bar: 10 µm. (D) lineage expression of deletion constructs generated from egl-13FL::GFP. Restriction sites utilized, approximate deletion lengths, and end points as distances from the translational start are indicated.

View larger version (52K): [in this window] [in a new window]   Fig. 2. LAG-1 and Fos/Jun cis binding elements are required for egl-13 expression at cell specification and for rescue of egl-13 mutant defects. (A) Conservation of transcription factor binding sites within the 1.3 kb enhancer of egl-13. Conserved cis-elements are shown in bold. Dark gray boxes represent sequences sharing remarkable identity with C. briggsae: 106 bp box 1 in elegans shares 88.2% identity with briggsae, 21 bp box 2 shares 100%, 65 bp box 3 shares 89.6%, 36 bp box 4 shares 94.4%, 142 bp box 5 shares 91.7%, and 45 bp box 6 shares 93.5%. Trans factor abbreviations (in order of relative upstream positions): Hb, Hunchback; Dof, Dof domain; MBF1, Multi-protein binding factor; Y-box, Y-box binding protein; Etsr, ETS domain related; HOM, homeodomain; POU, POU domain; HES, Hairy/Enhancer of split; TFIID, transcription factor II D; ETS domain; NBF, Nonamer binding protein; STRE, stress response element; ZnF, zinc finger; LIM, LIM domain; NF-B or Rel homology; Sp1 or ZnF; FOX, F-box/Forkhead; GATA, GATA binding factor; BR-C, Broad complex or ZnF; E2F factor; TBP, TATA binding protein; SRY, SRY or high mobility group (HMG). (B) Effect of LAG-1 or Fos/Jun deletion on temporal uterine egl-13FL::GFP fluorescence. DIC images showing medial uterine-vulval development. Lateral expression of egl-13FL::GFP in cells is seen here just after the cells have divided. Neither egl-13FLLAG-1::GFP nor egl-13FLFos/Jun::GFP transgenic animals fluoresce at specification. egl-13FLLAG-1::GFP expresses in all descendants later, whereas later expression of egl-13FLFos/Jun::GFP is only in uv1. Brackets encompass the cells. Dashed ovals surround uterine area where fluorescence is lost. Asterisks mark body wall expression. Scale bar: 10 µm. (C) Four constructs of egl-13FL::cDNA were generated with intact, singly, or doubly-deleted LAG-1 and Fos/Jun binding sites. L4 stage hermaphrodites from independent egl-13(ku194) transgenic lines for each construct were scored over 4 days for egg laying. Egl+ indicates healthy appearance and egg laying. Egl+/-indicates egg laying but bloated appearance and/or rupture from internal hatching. Egl-indicates no egg laying and eventual rupture. ku194 is 100% Egl-(n=100s, not shown). The strain designations are provided in Materials and methods.

View larger version (69K): [in this window] [in a new window]   Fig. 3. fos-1 loss-of-function causes abnormal uterine morphology and absence of uterine egl-13 expression. (A) Specification and mid L4 stage expression of egl-13::GFP(tyIs4) in the wild type and fos-1(ar105). fos-1(ar105) do not express GFP in uterine tissue (dashed ovals indicate where fluorescence was expected; fluorescent nuclei in the wild type are bracketed). The mid L4 stage uterus in ar105 is undifferentiated by comparison with the wild type, as apparent by the absence of a uterine lumen. Arrow indicates the utse, observed only in the wild type. Other GFP-positive cell bodies belong to the body wall, where fluorescence is not affected. (B) No uterine egl-13::GFP expression or lumen is observed after fos-1 RNAi treatment. Scale bar: 10 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 4. fos-1 function is required for uterine but dispensable for vulval expression of lin-11. (A-H) Comparison of late L3 stage lin-11::GFP(syIs80) expression in the wild type and fos-1(ar105). (A,B) In the medial plane (med) of the wild type, lin-11 expression is observed in the 2° vulval cells closest to the 1° vulval cells. (C,D) In the lateral plane (lat) of the wild type, out of the five VU intermediate precursors, lin-11 is expressed in the three cells. In the medial plane of fos-1, lin-11 is also expressed in 2° vulval cells (E,F); however, laterally, lin-11 expression is absent in the uterus (G,H). (I-R) Early to mid L4 stage expression of lin-11::GFP in the wild type and fos-1(ar105). 1° vulval cells, vulF and vulE, and 2° vulval cells, vulD, vulC, vulB2 and vulB1, are indicated. In the medial (I,J) and sub-lateral (sub-lat, K,L) planes of the wild type, terminal descendants of the 2° vulval lineage continue to express lin-11. Expression is also detected in descendants (L). In the medial (N,O) and sub-lateral (P,Q) planes of fos-1, lin-11 expression is appropriately detected in the 2° lineage. However, no uterine lin-11 expression is observed (Q). Black arrowheads point to the AC. White arrowheads point to uterine intermediate precursor cells. In A,B and E,F, brackets indicate vulval cells; elsewhere, brackets indicate cells. Dashed ovals surround uterine area where fluorescence is lost. White arrows point to GFP+ 2° vulval cell descendants. Asterisks mark expression in VC neurons. Scattered background signals are from gut autofluorescence. Scale bar: 10 µm.

View larger version (67K): [in this window] [in a new window]   Fig. 5. FOS-1 is expressed in the intermediate precursors and co-localizes with EGL-13 during the lineage. (A) fos-1 locus with isoform a, b, and putative c translational starts and origin of plastFOS-1c::YFP indicated. (B-F) Late L3 stage. (B) The AC (black arrowhead) is invading underlying primary vulval cells (black bracket). (D) Approximately 5 µm from B, FOS-1c is expressed in all dorsal and ventral uterine cells including the VU intermediate precursors (C, white arrowheads). (E) Three cells express egl-13::CFP (white bracket). (F) Merged image of D and E showing that FOS-1c co-localizes with egl-13 at specification. (G-J) Mid L4 stage. (H) FOS-1c is broadly expressed in numerous uterine cells. White arrows point to six cell daughters plus the AC. (I) descendants continue to express egl-13::CFP. (J) Merged image of H and I showing FOS-1c also co-localizes to the descendants. Asterisks mark non-specific fluorescence from the GFP co-transformation marker. Scale bar: 10 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 6. FOS-1 can directly bind to egl-13 URS as a heterodimer with JUN-1, which is also expressed throughout the uterus at cell specification. (A) EMSA with labeled 100 bp homologous template containing the Fos/Jun binding site. Left gel: No band shift was detected with only lysate present (, lane 1), FOS-1 alone (lane 2), or JUN-1 alone (lane 3). A shifted complex (bottom arrow) was observed in the presence of both FOS-1 and JUN-1 (lane 4) and also with a Myc-tagged version of FOS-1 with JUN-1 (lane 5). Addition of polyclonal anti-Myc antibody in lane 6 produced a supershift due to increase in molecular mass of DNA-protein complex plus Ab (top arrow) and also completely neutralized the original band shift (bottom arrow) due to titration of protein from forming a stable complex. Right gel: The supershift was significantly reduced in the presence of unlabeled (COLD) sequence containing the egl-13 Fos/Jun binding site (lanes 4-6) but not effectively reduced by an excess of unlabeled sequence carrying a mutated Fos/Jun binding site (lanes 7-9). Gray triangles above lanes 4-6 and 7-9 represent increasing amounts (5x, 25x and 125x, respectively) of unlabeled 30 bp competitor oligonucleotides. (B) This diagram represents the position of the isolated 5.3 kb genomic sequence in pJUN-d/c::GFP in the context of the entire jun-1 locus. The NruI site is a starting reference point for 5 kb upstream of the first transcript. The translational starts for verified isoforms are indicated. (C-F) Uterine JUN-1 expression. (C,D) In the medial plane, expression of pJUN-d/c::GFP is detected in the AC (black arrowhead) as well as throughout the dorsal and ventral uterus. (E,F) In a lateral plane, VU intermediate precursors (white arrowheads) also express the translational reporter. Scale bar: 10 µm.