1. Volume 9, Issue 6, Pages 745-756 (December 2005)
IFITM/Mil/Fragilis Family Proteins IFITM1 and IFITM3 Play Distinct Roles in Mouse Primordial Germ Cell Homing and Repulsion 
Satomi S. Tanaka, Yasuka L. Yamaguchi, Bonny Tsoi, Heiko Lickert, Patrick P.L. Tam 
Developmental Cell 
Volume 9, Issue 6, Pages (December 2005)
DOI: /j.devcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Expression Domains of Ifitm1 and the Location of PGCs
(A–G) Ifitm1 expression revealed by whole-mount in situ hybridization of embryos at the (A) E6.75 early-streak stage, (B) E7.0 mid-streak stage, (C) E7.25 mid- to late-streak stage, (D) E7.5 late-streak to early-bud stage, (E) E8.25 early-head-fold stage, (F) E somite stage, and (G) E –34 somite stage.
(H–M) PGCs revealed by alkaline phosphatase (AP) activity in (H) E7.25 mid-streak-stage, (I) E7.5 early- to late-bud-stage, and (J) E8.25 early-head-fold-stage embryos. Pou5f1 (Oct-3/4) expression in a (K) E9.0 embryo, and (L) Iftim1 and (M) AP expression in E10.0 embryos (the plane of sections in [L] and [M] is shown in [G]).
(N–R) The localization of PGCs in (N) E7.25 (see [H] for section orientation) and (O) E7.5 (see [I] for section orientation) relative to (P) the Ifitm1-expression domain in a E7.5 embryo (plane of the section shown in [D]), and the localization of PGCs in (Q–Q″) the posterior endoderm and allantois (plane of sections shown in [J]) relative to (R) the Ifitm1 expression domain in a E8.25 embryo (plane of the section shown in [E]).
Arrows indicate the cell cluster of PGC precursors. Arrowheads indicate PGCs. al, allantois; ect, ectoderm; en, endoderm; hg, embryonic hindgut; mes, mesoderm. Crossed arrows indicate the embryo orientation in (A)–(D), (H), and (I). (E) and (J) are posterior views. (F), (G), and (K) are right-side views. The scale bars represent 100 μm in (A)–(K) and 20 μm in (L)–(R).
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2
IFITM1 and IFITM3 Fusion Proteins Are Targeted to the Cell Surface
(A–D) Panels in column I show GFP expression from transfected vector in STO cells (green), those in column II show immunofluorescence of FLAG- or Myc-tagged fusion proteins (red), and those in column III show the merged image of the GFP signal and immunofluorescence. Panels in column IV show merged images viewed under the confocal microscope. (A and B) Localization of the IFITM1-FLAG protein and (C and D) IFITM3-Myc protein on the cell surface revealed by nonoverlapping signals. The cell surface localization of the Ifitm3-Myc protein agrees with the result of immunostaining with anti-IFITM3 antibody (Saitou et al., 2002; Matsui and Okamura, 2005).
(E–J) Localization of variant forms of tagged IFITM1 protein in the peripheral region of the cell: (E and F) truncated form containing the N-terminal extracellular domain and the first transmembrane domain (Iftim1(ΔC)-FLAG), (G and H) truncated form containing the two transmembrane and the C-terminal intracellular domains (Ifitm1(ΔN)-FLAG), and (I and J) chimeric protein containing the N-terminal extracellular domain of IFITM3 and the transmembrane domains and the C-terminal intracellular domain of IFITM1 (Ifitm3(N)+Ifitm1(C)-FLAG).
(K–L) Truncated protein containing only the N-terminal extracellular domain of Ifitm1 (Ifitm1(ΔTM-C)) widely distributed in the cell rather than localized to the cell surface as revealed by overlapping GFP and immunofluorescence signal (yellow-orange; [K″] and [L]). STO cells transfected with GFP expression vector showed an intense GFP signal and no immunofluorescence (data not shown). The scale bar represents 5 μm.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3
Ifitm1 Activity Influences the Distribution of PGCs in the Endoderm
(A) Electroporation of E7.5 embryos: orientation of the cathode needle (for focusing the electroporation) and the anode plate across the embryo.
(B and C) GFP-expressing cells in the endoderm after (B) 5 hr and (C) 24 hr of in vitro culture.
(D–D″) GFP-expressing endodermal cells (D) coexpressing the IFITM1-FLAG protein ([D′] IFITM1-FLAG fusion protein; [D″] merged image).
(E–E″) GFP-expressing cells intermingled with the DPPA3-positive PGCs in the posterior endoderm.
(F–F″) DPPA3-positive PGCs were excluded from sites in the posterior endoderm where Ifitm1 was expressed. (E and F) GFP-expressing cells; (E′ and F′) DPPA3-positive PGCs, and (E″ and F″) merged image. White arrowheads point to the region where PGCs are normally present but were excluded.
(G–L) Nonoverlapping distribution of AP-positive PGCs and GFP-expressing cells in the posterior endoderm. The left column panels show GFP-expressing cells, and the right column panels show AP-positive PGCs.
The black arrowheads point to the PGCs, and asterisks mark GFP-positive cells with Ifitm1 activity. The scale bar represents 10 μm in (D″) and 100 μm in other panels.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4
Activity of Variant Forms of IFITM1 and the Interaction with IFITM3 on the Pattern of Cell Distribution
(A–X) Exclusion of (A–A″) DPPA3-positive and (B and C) AP-positive PGCs from the domain of GFP-expressing cells in CMV-Ifitm1(ΔC)-transfected embryos, but not in embryos transfected with (D and E) CMV-Ifitm1(ΔTM-C), (F and G) CMV-Ifitm1(ΔN), or (H and I) CMV-Ifitm3(N)-Ifitm1(C) (arrows). (J and K) Cells expressing CMV-GFP in the control embryo are distributed widely in the posterior endoderm, but cells expressing (L–N) CMV-Ifitm3 or (O and P) CMV-Ifitm1+CMV-Ifitm3 were localized predominantly to the sites of AP-positive PGCs (arrows) and DPPA3-positive PGCs in the posterior endoderm. (A and L) GFP-expressing cells; (A′ and L′) DPPA3-positive PGCs, and (A″ and L″) merged image. White arrowheads in (A)–(A″) point to the region in the normal sites where PGCs were excluded. Black arrowheads in (C) mark the PGCs that were displaced the farthest from the Ifitm1(ΔC)-expressing cells (marked by asterisks). Arrows in other figures mark the PGCs that were found at the normal site. (Q and R) Ifitm3- and AP-positive PGCs in the crescent-shaped domain in the posterior endoderm of the early-head-fold-stage embryos. (S) CMV-Ifitm3, (T) CMV-Ifitm1+CMV-Ifitm3, and (U) CMV-Ifitm3(N)-Ifitm1(C)-expressing cells tended to localize to the PGC domain, whereas (V) CMV-GFP, (W) CMV-Ifitm1, and (X) CMV-Ifitm1(ΔC)-expressing cells were more widely distributed. The black arrowheads in (Q) and (R) point to the Ifitm3- and AP-expressing PGCs. The scale bar represents 100 μm.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5
PGCs in the Hindgut Are Responsive to Ifitm1 and Ifitm3 Activity
(A–C) (A) Electroporation of the vector into the posterior endoderm of a E8.25 embryo, followed by examination of (B) GFP-expressing and (C) AP-positive PGCs after 30 hr of in vitro culture.
(D–R″) (D–J′) AP-positive PGCs; (L′, P′, and R′) 4C9-positive and (N′) DPPA3-positive PGCs were excluded from the vector-expressing segment of the embryonic gut when the embryos were transfected with (G, G′, and K–L″) CMV-Ifitm1 or (H, H′, and M–N″) CMV-Ifitm1(ΔC), but not with (D, D′, and Q–R″) CMV-GFP, (E and E′) CMV-Ifitm3, (F and F′) CMV-Ifitm1+CMV-Ifitm3, (I, I′, and O–P″) CMV-Ifitm1(ΔN), or (J and J′) CMV-Ifitm3(N)-Ifitm1(C). GFP fluorescence and AP staining results are shown for (D)–(J′), and GFP, immunofluorescence, and merged images are shown for (K)–(R″). White arrowheads in (G) and (H) mark the vector-expressing cells, black arrowheads in (G′) and (H′) indicate the site where PGCs were absent, and the boxed area in (K), (M), (O), and (Q) marks the region of the specimen that was visualized by immunofluorescence. The scale bar represents 100 μm.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6
Knockdown of Ifitm1 Activity by shRNA Leads to Ectopic Localization of the PGCs
(A and A′) (A) A control R1 ES cell-derived E8.5 embryo showing (A′) localization of Dppa3-positive PGCs in the posterior gut endoderm.
(B and B′) (B) A shRNA knockdown ES cell-derived E8.5 embryo showing (B′) ectopic localization of the PGCs in the posterior mesoderm away from the normal site in the posterior gut endoderm, with a similar number of PGCs as the control embryo.
(C and C′) (C) A shRNA knockdown ES cell-derived E8.5 embryo showing (C′) localization of the PGCs exclusively in the posterior mesoderm and allantoic mesoderm.
Arrows indicate PGCs at the normal site in the hindgut endoderm, and arrowheads mark PGCs in the ectopic site. (A) and (B) show a posterior view; (C) shows a lateral view. Dotted lines in (A)–(C) indicate the plane of the histological section in (A′)–(C′). al, allantois; am, aminion; en, endoderm; mes, posterior mesoderm. The scale bar represents 20 μm.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7 Ifitm Gene Activity and Movement of the PGCs
(A) In the E7.0 embryo, PGC precursors displaying Ifitm1 and -3 activities are localized in a cluster within the Ifitm1-expressing tissue in the posterior primitive streak.
(B) In the E7.25 embryo, downregulation of Ifitm1 activity in the PGC precursors accompanies the segregation of the presumptive PGCs. The movement of Ifitm1-negative PGCs to the endoderm (yellow arrow) may be mediated by the repulsive activity (green block-arrow) of the Ifitm1-expressing somatic cells of the primitive streak, resulting in their localization in the endoderm where Ifitm1 is not expressed. The movement of the PGCs in the endoderm may be guided by the Ifitm3 activity, which leads to the homing of PGC domains to the posterior region of the gut endoderm (red arrow). In essence, Ifitm activity may navigate PGCs to the posterior endoderm by repelling them from the Ifitm1-expressing primitive streak tissue, while Ifitm3 activity in PGCs elicits the responsiveness to some uncharacterized guidance cue for homing in the endoderm.
(C) In the E8.25 embryo, PGCs are confined to the Ifitm1-free endoderm by their continuous response to the repulsive activity (green block-arrow) of the adjacent Ifitm1-expressing mesoderm as they are incorporated into the hindgut invagination and then distributed along the length of the embryonic gut (red arrow).
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions