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First published online 10 January 2007
doi: 10.1242/dev.02769

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C. elegans EVI1 proto-oncogene, EGL-43, is necessary for Notch-mediated cell fate specification and regulates cell invasion

Howard Hughes Medical Institute and Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125, USA.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Characterization of the enhancer that drives LIN-3 expression in the pre-AC/pre-VU and AC. (A) In wild-type animals, Z1.ppa and Z4.aap cells always become VU cells and Z1.ppp and Z4.aaa cells have equal potential to become the AC or a VU. (B-D) In wild-type animals, LIN-3::GFP is expressed (B) in the pre-AC/pre-VU cells before AC/VU specification and (C) in the AC after the specification. Multiple gonadal cells express LIN-3::GFP after specification in the lin-12 (null) background (D). LIN-3::GFP is not expressed in any gonadal cells of the lin-12 (gain-of-function) animals after mid-L2 stage where all four pre-AC/pre-VU cells are transformed into VU cells (data not shown). (E) Anchor Cell-specific Enhancer of LIN-3 (ACEL) is sufficient to express GFP in both AC and pre-AC/pre-VU cells. Site-directed mutations of the E-boxes or FtzF1 NHR binding site, but not those in other regions, eliminate GFP expression in both cell types. Expression of these constructs in the AC was reported previously (Hwang and Sternberg, 2004).

View larger version (67K): [in this window] [in a new window]   Fig. 2. EGL-43 is required for AC/VU specification and AC invasion. Images of the gonad and vulva region of hermaphrodites in the mid-L3 (2 and 4-cell P6.p stage; A-F,I,J) and the early L4 (8-cell P6.p stage; G,H,K-N) stages. (A-H) Nomarski (left), fluorescence (right) and overlay (center). (I-N) Nomarski (top), fluorescence (bottom) and overlay (middle). Transgenic animals that contain (A,B) lin-3::GFP (syIs107, for the AC), (C,D) cdh-3::GFP (syIs50, for the AC), (E,F) lag-2::YFP (syIs128, for the DTC), or (M-N) sparc::GFP (syIs113, for the basement membrane) were soaked in the control or egl-43 dsRNA solution. (G) In control RNAi-treated animals that carry a gain-of-function mutation (n137) of lin-12, the AC is transformed into a VU cell. Additional vulval cells (Muv phenotype) express cdh-3::GFP (green) at early L4. (H) In animals treated with egl-43 RNAi, two ACs expressing cdh-3::GFP (green, arrows) are present owing to the suppression of activated LIN-12 during AC/VU specification. However, egl-43 RNAi does not suppress the Muv phenotype caused by the activated LIN-12. (I) With control RNAi, the basement membranes are interrupted (between the arrowheads) underneath the AC (expressing cdh-3::GFP in white), and the basolateral side of the AC crosses through the hole in the basement membranes and penetrates between central 1°-fated vulval cells. (J) In animals treated with egl-43 RNAi, two partially overlapping ACs (expressing cdh-3::GFP in white) cannot cross the unbroken basement membranes. A distinct line representing the juxtaposed gonadal and ventral epidermal basement membranes is visible under Nomarski optics, separating the AC from the P6.p granddaughters. (K) The basolateral portion of the AC invades between the central P6.pap and P6.ppa cells. Because some vulval cells also express cdh-3:GFP at early L4, the cytoplasmic GFP signals in the AC (arrow) and vulval cells overlap. (L) In animals treated with egl-43 RNAi, the GFP signal in the ACs is separated from those in the vulval cells because the ACs (arrows) could not invade into the vulval epithelium. The basement membranes between gonad and vulval epithelium are (N) intact in the egl-43 RNAi or (M) lost (between the arrowheads) in the control RNAi underneath the AC (arrow) when viewed by sparc::GFP (white).

View larger version (79K): [in this window] [in a new window]   Fig. 3. Site-directed mutagenesis of E-boxes of the ACEL-like element and LAG-1 binding sites in egl-43. (A) egl-43 gene structure. The ACEL-like element that contains two E-boxes is predicted in the first intron, and eight LAG-1 binding sites are predicted in the 5' regulatory and the first intronic regions. (B,C) egl-43 (wild type)::YFP is expressed (green) at early L2 in the pre-AC/pre-VU cells located in two different focal planes. (D,E) Either the Z1.ppp or Z4.aaa cell moves to the central position on the ventral surface of the gonad to become the AC (green, arrow), which is located (D) in a focal plane different from the two focal planes containing the three VU cells (green). Only one VU focal plane is shown in E. (F,G) The AC begins to express cdh-3::CFP at late L2. The AC (arrow) is appears yellow in F because of the co-localization of cdh-3::CFP (red) and egl-43::YFP (green). (H,I) The egl-43 (mutated E-boxes)::YFP is not expressed in the pre-AC/pre-VU cells, AC and VU cells until late L2. (J,K) egl-43 (mutated E-boxes)::YFP is expressed in the three VU cells (green), and not in the AC (arrow), when the cdh-3::CFP begins to be expressed in the AC (J, red) at late L2. (L-Q) The egl-43 (mutated LAG-1 sites)::YFP is expressed in the pre-AC/pre-VU cells at early L2 (L,M, green). One cell (Z4.aap) expresses YFP very weakly in this animal (L). At mid-L2, YFP expression is retained in the AC (N, in green, arrow), but is lost or decreased in the VU cells (O). At late L2 when the cdh-3::CFP (red) begins to be expressed in the AC (P, arrow), three VU cells regain the YFP signal (Q). The AC expresses both cdh-3::CFP (red) and egl-43 (mutated LAG-1 sites)::YFP (green) at this stage, resulting in the merged yellow colour (P). The egl-43 (mutated LAG-1 sites)::YFP (green) begins to be expressed in two DU cells (yellow arrow) right after the three VU cells regain YFP expression (Q). Only one VU focal plane is shown. For each construct, about 100 animals were examined from six to ten transgenic lines. The pictures represent typical expression patterns at specific stages. The lines include syEx895 and syEx896 for the wild type; syEx891 and syEx892 for the mutated E-boxes; syEx897, syEx898, syEx899 and syEx900 for the mutated LAG-1 binding sites.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Multiple regulatory elements are required for EGL-43 expression in the AC, DU and VU lineages. (A) Results of the site-directed mutagenesis study and fos-1 RNAi are summarized. White areas represent the absence of egl-43::YFP expression. Green areas represent the presence of YFP expression at specific developmental stages. The areas marked in light green represent decreased egl-43::YFP expression. (B) Model summarizing egl-43::YFP expression patterns in the somatic gonad. The ACEL-like element containing two E-boxes is the major enhancer expressing EGL-43 in the pre-AC/pre-VU cells and in the AC, but not in the VU cells. LAG-1 binding sites are also required for EGL-43 expression in the pre-VU cells when AC/VU cell fates become specified. Unidentified enhancers increase EGL-43 expression in the VU cells and their descendants from late L2, and in the AC from mid-L3.

View larger version (33K): [in this window] [in a new window]   Fig. 5. EGL-43 is involved in AC invasion as a downstream target of HLH-2 and FOS-1. Animals containing cdh-3::CFP (red) were injected with egl-43 (PR-domain)::YFP constructs. (A-F) The PR-YFP transgene is expressed (green) in the VU and DU lineages as well as in the AC from early L2 when using the wild-type egl-43 regulatory region (A,B), and shows the AC (cdh-3::CFP, white) invasion defect (E). The transgene is expressed in the VU and DU lineages from late L2, but in the AC from mid-L3, when using the mutated E-boxes (C,D), and shows normal AC invasion (F). (G-L) egl-43 RNAi eliminates expression of zmp-1::GFP (G,H) and him-4::GFP (I,J) in the AC (arrow), but not of fos-1::YFP (K,L). egl-43 RNAi resulted in two ACs in which fos-1::YFP is expressed higher than in the neighboring gonadal cells (L, arrows). (M-R) fos-1 RNAi greatly reduces egl-43 expression in the AC, and in the DU and VU descendants at the L3 molt (M,N) and at mid-L3 (Q,R), but not at mid-L2 (O,P). Animals from a transgenic line that expresses egl-43(exon-5)::YFP were used for the fos-1 RNAi.

View larger version (30K): [in this window] [in a new window]   Fig. 6. HLH-2 and LAG-1 bind to egl-43 regulatory regions. (A,B) EMSA with in vitro translated (TNT) LAG-1. Shifted complexes (B1 and B2) appear in the presence of LAG-1. The binding complexes are competed by DNAs containing the wild-type (w) but not the mutated (m) LAG-1 binding sites. `F' indicates the migration of the free DNA probe. (C,D) EMSA with purified HLH-2. A shifted complex `B' appears with HLH-2 and the wild-type ACEL-like probe, but not with Luciferase or with the probe on which both E-boxes are mutated. (D) The binding complex is competed by DNAs containing the wild-type (w) but not the mutated (m) E-boxes. DNA sequences are listed in Table 1; the numbers in B and D correspond to those of DNA competitors in Table 1. (E) PCR analysis from ChIPs performed with the extracts from animals expressing SEL-8::GFP. Pre-IP represents the input extracts subjected to IP. The 5'-region of lip-1, which contains four LAG-1 binding sites and was shown to be immunoprecipitated with LAG-3 (SEL-8) antibodies (Lee et al., 2006), was used as a positive control.

View larger version (13K): [in this window] [in a new window]   Fig. 7. EGL-43 and HLH-2 are required for AC/VU specification, VPC proliferation and AC invasion. (A) egl-43, fos-1, him-4, hlh-2 and zmp-1 form a regulatory network necessary for the AC to invade vulval epithelium. (B) hlh-2 is involved in AC/VU specification by expressing lag-2 and egl-43. The same type of enhancer (ACEL) is responsible for the expression of lin-3 and egl-43 in the AC, which are essential for the induction of VPCs and AC invasion.

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