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First published online 14 November 2007
doi: 10.1242/dev.008680

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Notch-GATA synergy promotes endoderm-specific expression of ref-1 in C. elegans

Alexandre Neves^1,2,*, Kathryn English^1,* and James R. Priess^1,3,

¹ Howard Hughes Medical Institute, Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
² Gulbenkian PhD Programme in Biomedicine, Rua da Quinta Grande, 6, 2780-156, Oeiras, Portugal.
³ Department of Biology, University of Washington, Seattle, WA 98195, USA.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Control of Notch-dependent endoderm expression. (A) Diagram of the two Notch interactions in the endoderm. Cells in the endoderm primordium that are activated by Notch signaling are shown in green. (B-G) The top and bottom panels show embryos at the E8 and E16 stages, respectively, with anterior at top. Images were taken through the plane of the intestinal primordium (outlined in black); the E16 primordium appears smaller than the E8 because the posterior end curves downward, out of the focal plane. (B) Differential interference contrast (DIC) micrographs. (C-F) Fluorescence micrographs of embryos with wild-type or mutant enhA::GFP transgenes; mutations were introduced into either pAN20 or pKG55 (Fig. 2A). (C) Wild-type enhA::GFP (pAN20); the onset and pattern of GFP expression in the endoderm is identical to ref-1::REF-1::GFP expression described previously (Neves and Priess, 2005). (D) CSL sites mutated to RAGGCAA (pAN20). (E) WGATAR sequences mutated to WCATAR (pKG55). (F) Candidate NK site mutated to CTCAAGTT (pAN20). (G) Wild-type embryos depleted for ELT-2/GATA by RNAi (GFP reporter is ANPCR in Fig. 2A).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Identification of a Notch-regulated endoderm/mesoderm enhancer. (A) The ref-1 promoter regions shown were fused to GFP and assayed for expression in Notch-activated endodermal cells (green, expressed; red, not expressed). Transgenes were generated as plasmids (e.g. pAN20) or PCR products (e.g. ANPCR); pAN2 and ANPCR have been described previously (Neves and Priess, 2005). Numbers indicate distance from the initiator ATG (position 0). Inverted black and white triangles represent CSL- and GATA-binding sites, respectively. (B) Diagram of C. elegans enhA. Cyan and yellow arrows indicate orientation of CSL and GATA sites, respectively. The green box represents a possible NK-binding site. Bold line indicates region used for EMSA experiments (see Fig. 4). (C) Sequence alignment of the enhA element from C. elegans (C.e), C. remanei (C.r) C. briggsae (C.bri) and C. brenneri (C.bre). Black lettering indicates that the nucleotide is conserved in at least three species. Note that the 3-CSL sequence in C. brenneri (TCTGGGAA) differs from a consensus CSL binding sites (YRTGRGAA) (Yoo et al., 2004); this sequence was confirmed by PCR amplification and analysis of genomic DNA from C. brenneri.

View larger version (90K): [in this window] [in a new window]   Fig. 3. Notch-regulated gene expression in pm8. (A) DIC image of wild-type embryo at the 1.5-fold stage focused at the plane of the pharyngeal primordium (bracket); anterior is left. (B) Same embryo as in A, showing GFP expression from enhA::GFP (pKG55). The bent line indicates a pair of sister cells in the pharynx that express ref-1 independent of Notch signaling; these cells are below the focal plane shown, and appear faint. Asterisks here and in panels D and E indicate intestinal cells with faint GFP expression persisting from the second endodermal Notch interaction. (C,D) DIC and fluorescence image of 1.5-fold stage embryo showing C. briggsae enhA::GFP expression. (E) GFP expression after mutating the potential NK site in enhA to CTGTGCTGC. (F) GFP expression after mutating all WGATAR sequences in enhA to WCATAR. (G,H) DIC and fluorescence images of a 3-fold stage embryo showing enhA::GFP expression in pm8. Small spots appearing posterior to the pharynx in H and below in J are autofluorescent granules in the intestine not related to transgene expression. (I) Lack of GFP expression in pm8 after mutating the NK site to CTCAAGTTC. (J) Expression of the same transgene used for I after heat-shock expression of ELT-2/GATA.

View larger version (64K): [in this window] [in a new window]   Fig. 4. In vitro binding of LAG-1/CSL and ELT-2/GATA to enhA sequences. (A) Electrophoretic mobility shift assay (EMSA) using GST::LAG-1 and a previously described probe that binds LAG-1 through two CSL-binding sites (Hwang et al., 2007); arrowhead indicates free probe. Competitor DNA is from enhA (see Fig. 2B). 2-CSL and 3-CSL indicate wild-type sites; 2-CSL* and 3-CSL* are ATGGGAA to AAGGCAA and GTGGGAA to GAGGCAA mutations, respectively. (B) EMSA using in vitro translated ELT-2 and a labeled probe from the pho-1 endodermal enhancer (Fukushige et al., 2003). Competitor DNA is the same as in A. 2-GATA and 3-GATA are the wild-type sites; 2-GATA* and 3-GATA* are mutations from GATA to CATA. Note that competition with the [2-GATA 3-GATA*] probe is approximately fivefold less than with the [2-GATA* 3-GATA] probe.

View larger version (74K): [in this window] [in a new window]   Fig. 5. ELT-2/GATA is sufficient for enhA::GFP expression in non-endodermal cells activated by Notch. (A) Expression of a ref-1 family member, hlh-26, following Notch interactions in AB descendants. The embryo is at the 26-cell stage, anterior to left. Cells labeled 1 are AB descendants activated by Notch signaling beginning at the four-cell stage of embryogenesis; cells labeled 2 are AB descendants activated by Notch signaling beginning at the 12-cell stage of embryogenesis. The arrow indicates an AB descendant that does not contact any ligand-expressing cells, and so is not activated by either the first or second interaction. The asterisk here and below indicates the P4 blastomere; this blastomere is transcriptionally quiescent, and thus serves as a negative control for expression levels. (B) Lack of enhA::GFP expression in AB descendants in wild-type embryos. Here and below, the presence of the transgene was confirmed by either Notch-dependent or Notch-independent expression at later embryonic stages. (C-H) DIC and fluorescent images of similarly oriented embryos at the same developmental stages following heat shock of ELT-2/GATA. Because cell division patterns are invariant in early C. elegans embryos, the Notch-activated AB descendants (sets 1 and 2) can be identified solely by position. (E,F) Lack of GFP expression after mutating all WGATAR sequences to WCATAR. (G,H) Lack of GFP expression after mutating all CSL sites to RAGGCAA.

View larger version (34K): [in this window] [in a new window]   Fig. 6. LAG-1/CSL and ELT-2/GATA bind directly in vitro. Full-length or truncated 35S-labeled ELT-2/GATA was tested for binding GST-LAG-1(1-674) and GST-LAG-1(192-663). Negative controls are GST alone and a GST-fusion to a cadherin domain (see Materials and methods).

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