Ecsit expression in wild-type embryos as revealed by whole-mount in situ hybridization with Ecsit2-specific probes. (A) E6.25 embryos hybridized with (left) antisense probe or (right) sense probe. (B) Sagittal section of the left embryo in A. Note expression in epiblast and extraembryonic ectoderm. (C) E7.5 embryo hybridized with sense probe. (D-F) E7.0-E7.75 embryos hybridized with antisense probe. (G,H) Anterior (G) and posterior (H) view of an E7.5 embryo hybridized with antisense probe. (I-K) Cross-sections of the embryo in H along dashed lines. The color was developed for 200 h in A, 17 h in C-F, and 90 h in G and H. (exe) Extraembryonic ectoderm; (ve) visceral endoderm.

Expression of developmental markers in Ecsit mutant embryos. Whole-mount in situ hybridization with antisense probes of indicated genes. In all the panels, anterior faces to the left when anterior-posterior orientation can be identified. A minimum of five mutant embryos were assessed for each probe at each developmental time point. (Normal) Ecsit^+/+ or Ecsit^+/-; (Mutant) Ecsit^-/-. Bar, 300 μm.

Ecsit^-/- ES cells are compromised in their ability to contribute to mesodermal and endodermal derivatives in diploid aggregation chimeras. ROSA26 morulae were aggregated with either (A-C,H-J) Ecsit^+/+ ES cells (Ecsit^+/+ ↔ ROSA26) or (D-G,K-Q) Ecsit^-/- ES cells (Ecsit^-/- ↔ ROSA26). Embryos were dissected at either (A-G) E9.5 or (H-Q) E14.5, and the whole-mount embryos were stained for β-galactosidase, resulting in blue staining of ROSA26 cells. Counterstaining leads to red staining of Ecsit cells. Whereas Ecsit^+/+ ES cells were able to contribute to the embryos to various degrees at E9.5 (A: 100% contribution; B: ∼70% contribution; C: <20% contribution), embryos resulting from aggregations of Ecsit^-/- ES cells to ROSA26 morulae always showed very little contribution of the Ecsit^-/- cells at E9.5 (D; n = 14). Sectioning of these Ecsit^-/- ↔ ROSA26 chimeric embryos showed a large contribution of blue-stained ROSA26 cells in almost all tissues. Ecsit^-/- cells were able to contribute in a limited fashion to the neural tube (arrows in E), the dermatome of the somites (arrowheads in E and F), and the myocardium of the heart (arrowhead in G). At E14.5, Ecsit^+/+ cells were again able to contribute to the chimeric embryos to various degrees (H-J), whereas Ecsit^-/- ↔ ROSA26 chimeras always stained dark blue (K; n = 9). (L-Q) Sections of Ecsit^-/- ↔ ROSA26 chimeras. (L) Ecsit^-/- cells can efficiently contribute to neuroectoderm. (mb) Midbrain; (hb) hindrain; (arrow) choroid plexus. (M) Ecsit^-/- cells contribute efficiently to the neural tube (nt), the dermis (d), and epidermis (e), but not muscle tissue (m). (N) Limited contribution of Ecsit^-/- cells to heart tissue (arrowhead). (O) Low contribution of Ecsit^-/- cells to the villi (v) and smooth muscle (m) and efficient contribution to the mucosa (muc) in the gastrointestinal tract. (P) Limited contribution of Ecsit^-/- cells to embryonic liver. (Q) Very limited contribution of Ecsit^-/- cells to lung mesenchyme (arrowhead) and efficient contribution to lung epithelium (arrow).

Ecsit ShRNA inhibits Toll but not TNF signaling. Luciferase reporters containing two NF-κB binding sites (pBIIx-Luc) were cotransfected with pBluescript KS+ (Vector, 1:7 ratio), Sh1 (1:7), or Sh2 (1:1, 1:3, 1:7) into RAW 264.7 (A) or NIH/3T3 (B) cells. Then, 48 h after transfection, cells were treated (black bars) or untreated (open bars) with 100 ng/mL LPS (A) or 25 ng/mL TNFα (B) for 4 h prior to lysis and analyzed for luciferase activity.

Generation of heterozygous and homozygous Ecsit mutant ES cell lines. (A) Gene structure of the Ecsit locus. In Ecsit1, exon 5 is used in different frame from that in Ecsit2 and Ecsit3, as indicated by a black box in Ecsit1 instead of the open boxes in Ecsit2 and Ecsit3. (B) Targeting of the Ecsit locus. The targeting vector is described in Materials and Methods. Probe B and Probe C are indicated by bars. (B) BamHI; (X) XbaI. (C) Southern blot analysis of targeted ES clones. Using Probe C and BamHI digestion, the wild-type and targeted loci generate 6.2-kb and 3.2-kb bands, respectively. Using Probe B and XbaI digestion, the wild-type and targeted loci generate 18-kb and 3.1-kb bands, respectively. (D) Southern blot and Western blot analysis of Ecsit^-/- ES clones. The Southern blot was done by using Probe C and BamHI digestion, and 40 μg of whole cell lysates for each clone was used in immunoblotting (IB).

Histological analysis of Ecsit mutant embryos. (A-E) Wild-type (Ecsit^+/+) or heterozygous (Ecsit^+/-) embryos (A,C) and homozygous null (Ecsit^-/-) embryos (B,D,E) were embedded, sectioned, and stained with hematoxylin and eosin. Adjacent sections were hybridized with the Ecsit2-specific probe to genotype the embryos. Asterisk (^*) in D marks delaminating epiblast. In all panels, anterior faces to the left when anterior-posterior orientation can be identified. (al) Allantois; (am) amnion; (ch) chorion; (ec) extraplacental cone; (exe) extraembryonic ectoderm; (hf) head fold; (pe&rm) parietal endoderm and Reichert's membrane; (ps) primitive streak; (ve) visceral endoderm; and (vys) visceral yolk sac. Bar, 250 μm. (F) E6.75 embryo sections were stained with anti-phosphohistone H3 antibody (pH3) and counterstained with propidium iodide (PI). (G) Numbers of pH3-positive cells in two wild-type (wt1 and wt2) and two Ecsit^-/- mutant embryos (mut1 and mut2). (H) Whole-mount TUNEL staining of E6.75 embryos.

Escit^-/- ES cells are deficient in both proliferation and differentiation. (A) Growth curve of wild-type (Ecsit^+/+) and mutant (Ecsit^-/-) ES cells on feeder cells (feeders) and gelatin-coated plates (gelatin). Assays were done with two wild-type, one heterozygous, and six mutant ES cell lines; results from one wild-type and one mutant cell line are presented. The heterozygous cell line gave the same result as the wild-type cell lines. (B) Wild-type (Ecsit^+/+) and mutant (Ecsit^-/-) ES cells were injected subcutaneously into nude mice. The mice were photographed 7 wk after injection. (C) Histological analysis of teratomas derived from ES cells. Tissues arising from wild-type ES cells are shown in a, b, d, e, g, and h. Tissues arising from mutant ES cells are shown in c, f, and i. (ae) Adenoepithelium; (ael) adenoepithelium-like tissue; (bo) bone; (bm) bone marrow; (ca) cartilage; (iae) intestine adenoepithelium; (ke) keratinized epithelium; (mu) muscle; (ne) neural tissue; (nel) neural epithelium-like tissue; and (pc) Paneth cells.

Ecsit is essential for Bmp signaling. (A) Expression of Bmp4, Bmpr1a, and Tlx2 in mutant embryos. Whole-mount in situ hybridization was done with antisense probes of indicated genes. Anterior faces left when anterior-posterior orientation can be identified in all panels except g, in which the posterior view is presented. Bar, 300 μm. (B) Ecsit ShRNA inhibits Bmp signaling. (Upper panel) Flag-Ecsit2 and Ecsit ShRNAs were cotransfected into COS1 cells at 1:7 ratio. Then, 48 h after transfection, cell were lysed and whole cell lysates were immunoblotted with antibodies specific for Flag and GAPDH. (Lower panel) Luciferase reporters containing the promoter region of the mouse Tlx2 gene (Tlx2-Luc) were cotransfected with pBluescript KS+ (Vector, 1:7 ratio), Sh1 (1:7), or Sh2 (1:1, 1:3, 1:7) into P19 cells. Then, 48 h after transfection, cells were treated (black bars) or untreated (open bars) with 20 ng/mL Bmp4 for 15 h prior to lysis and then analyzed for luciferase activity. (C) Ecsit associates with Smad1 and Smad4. P19 cells were untreated or treated with 20 ng/mL Bmp4 for 30 min. Proteins precipitated (IP) with Ecsit2-specific antibody or preimmune serum (Pre.) were immunoblotted (IB) with antibodies specific for Smad1 and Smad4. The same amount of whole cell lysates (WCL) from each sample was immunoblotted with Ecsit2-, Smad1-, and Smad4-specific antibodies. (NS) Nonspecific; (IgH) immunoglobulin heavy chain. (D) Tlx2 promoter analysis. pGL2-Basic containing 5′ deletions of the Tlx2 promoter or pGL2-Promoter containing fragments spanning the Bmp-response element were transfected into P19. Then, 12 h after transfection, cells were treated with 20 ng/mL Bmp4 for 15 h prior to lysis and analyzed for luciferase activity. (E) Ecsit 2, Smad1, and Smad4 bind to the Tlx2 promoter. Soluble chromatin was prepared from P19 cells untreated or treated with 20 ng/mL Bmp4 for 30 min and immunoprecipitated with specific antibodies. The final DNA extractions were amplified using primers that cover the Tlx2 BRE and a 3′-region of the Tlx2 gene (4504/4759; Tlx2 3′).

Ecsit is a cofactor for Smad proteins in Bmp signaling. In unstimulated cells, Ecsit2 and Smad4 bind to the promoters of target genes, but are not able to drive transcription. Upon Bmp stimulation, Smad1 is phosphorylated, translocates to the nucleus, and forms a complex with Smad4 and Ecsit2 on the target gene promoters, which is then able to drive transcription. The red arrow indicates phosphorylation of the type I receptor by the type II receptor. (P) Phosphate group.