1. Two-Step Regulation of Left–Right Asymmetric Expression of Pitx2
Hidetaka Shiratori, Rui Sakuma, Minoru Watanabe, Hiromi Hashiguchi, Kyoko Mochida, Yasuo Sakai, Jinsuke Nishino, Yukio Saijoh, Malcolm Whitman, Hiroshi Hamada 
Molecular Cell 
Volume 7, Issue 1, Pages (January 2001)
DOI: /S (01)

2. Figure 1
Location of a Left Side–Specific Enhancer (ASE) in Intron 5 of Pitx2
(A) Various lacZ constructs containing different regions of mouse Pitx2 are shown below a representation of the genomic structure of the gene. Exons (1, 2, 3, 1c, 4, and 5) and two transcriptional initiation sites (P1 and P2) are indicated by closed boxes and open circles, respectively. The expression data for the various constructs in E8.2 mouse embryos are summarized on the right. The three numbers in parentheses correspond to the number of embryos showing asymmetric expression typical of Pitx2, the number of embryos exhibiting only ectopic expression, and the number of transgene-positive embryos showing no lacZ expression, respectively. FBA, first branchial arch; PAM, paraxial mesoderm.
(B) X-gal staining of E8.2 embryos harboring constructs 17-P1, NB7.0-P1, or 0.6-P1. Left–right (L–R) and anteroposterior (A-P) axes are indicated. X-gal staining is apparent in the left LPM including the splanchnopleure (sp) and somatopleure (so), as also revealed by the section taken at the level indicated by the line for the embryo expressing 0.6-P1. Scale bars are 500 μm.
Molecular Cell 2001 7, DOI: ( /S (01) )

3. Figure 2
Late-Stage Asymmetric Gene Expression Induced by the Pitx2 ASE
(A–D) Transgenic embryos harboring the 0.9-P1 construct were recovered at E10.5, stained with X-gal, and serially sectioned. Representative transverse sections are shown in (A)–(D). Broken lines indicate the midline. The construct 17-P1 gave rise to a similar expression pattern with the exception that it also exhibited bilateral staining in additional structures such as the branchial arch. bw, body wall; cac, common atrial chamber; ccv, common cardinal vein; dm, dorsal mesentery of the gut; lb, lung bud; pc, pericardiac cavity; st, septum transversum; and uv, umbilical vein. Scale bar is 500 μm.
(E) The X-gal staining patterns of other constructs were similarly examined and are summarized. +, asymmetric expression; −, no expression; and ×, bilateral expression. The relative level of asymmetric expression is indicated by the number of plus symbols. An asterisk indicates weak asymmetric expression in a small domain of the common atrial chamber. Multiple embryos were sectioned for most of the constructs, with each row representing an individual embryo. Embryos obtained from permanent transgenic lines are indicated by closed circles.
Molecular Cell 2001 7, DOI: ( /S (01) )

4. Figure 3
Pitx2 ASE Is Essential and Sufficient for Asymmetric Expression at Early and Late Stages
(A) Structure of three Pitx2-lacZ constructs. The solid blue circle indicates the hsp68 promoter.
(B–F) X-gal staining of E8.2 embryos harboring the indicated lacZ constructs. The 17-P1-ΔASE construct failed to induce L–R asymmetric expression in LPM but induced expression in the first branchial arch (F). Scale bar is 1000 μm.
(G–K) X-gal staining of E10.5 embryos harboring the indicated lacZ constructs. Transverse sections from similar levels are shown. L–R asymmetric expression is apparent with 0.6-P1(G) and 0.6-hsp (I) but not with lefty2 ASE-P1 (H), lefty2 ASE-hsp (J), or 17-P1-ΔASE (K). Open and closed arrowheads in (I) indicate asymmetric X-gal staining in the common atrial chamber and in the dorsal mesentery of gut, respectively. Scale bar is 1000 μm.
Molecular Cell 2001 7, DOI: ( /S (01) )

5. Figure 4 Structure and Function of FAST Binding Sites in Pitx2 ASE
(A) Organization of FAST binding sites (circles) in mouse Pitx2, ASE nodal ASE, and lefty2 ASE. Arrows in each circle indicate the orientation of the binding site.
(B) Nucleotide sequence of Pitx2 ASE (0.9 kb region). The three FAST binding sites (f1, f2, and f3) are shown in red, and an Nkx2-5 binding site is shown in blue. The 503, 502, and 302 deletion boundaries (see Figure 6A) are indicated.
(C) Luciferase reporter assay of Pitx2 ASE activity in MV1-Lu cells. Cells were transfected with luciferase reporter plasmids containing the indicated Pitx2 ASE sequences in the absence or presence of a FAST expression vector or the indicated amounts (micrograms) of an mSna expression vector. The transfected cells were incubated with or without TGF-β (2 ng/ml) for 12 hr, after which luciferase activity in cell lysates was measured. Data are expressed relative to the luciferase activity of unstimulated cells transfected with reporter construct alone.
(D) Luciferase reporter assay with the (f1)6-luc plasmid injected into Xenopus embryos. The reporter construct was introduced into embryos in the absence or presence of mRNAs encoding the indicated proteins. Luciferase activity was subsequently measured in animal caps. Data are expressed relative to the luciferase activity of embryos injected with reporter construct alone.
(E) Pitx2 gene is directly induced by FAST-1 or activin in Xenopus animal cap explants. Capped, in vitro transcribed FAST-VP16-GR RNA was injected to the animal pole region at 2-cell stage embryos, and animal cap cells were cut at blastrula stage. These animal cap explants were cultured with or without cycloheximide (CHX), dexamethasone (DEX), or activin protein until gastrula stage, and then endogenous Pitx2 gene expression was studied by PT-PCR analysis. EF1α was used for loading control. +RT and −RT represent the RT-PCR products from whole embryonic RNA that served positive and negative controls.
Molecular Cell 2001 7, DOI: ( /S (01) )

6. Figure 6
The Nkx2-5 Binding Site in ASE Is Essential for Late-Stage Pitx2 Expression
(A) Structure of various lacZ constructs of Pitx2. Red circles and the blue oval indicate binding sites for FAST and Nkx2-5, respectively.
(B and C) X-gal staining of E8.2 embryos harboring 0.9-P1 (B) or 0.9Nm-P1 (C). The two constructs induced indistinguishable patterns of L–R asymmetric expression in left LPM. Scale bars are 500 μm.
(D–I) Patterns of X-gal staining in E10.5 embryos harboring the indicated constructs. The staining pattern for hsp (I) was obtained with a stable line (22H) harboring this transgene; this line showed left-sided transgene expression in LPM at E8.2 (data not shown) but failed to show asymmetric expression at E10.5 (I). Scale bar is 500 μm.
Molecular Cell 2001 7, DOI: ( /S (01) )

7. Figure 5
FAST Binding Sites of Pitx2 ASE Are Sufficient for the Induction but Insufficient for the Maintenance of Pitx2 Expression
The expression patterns of the lacZ transgenes 0.9-P1 (A and D), 0.9Fm123-P1 (B and E), and (f1)6-hsp (C and F) were examined at both E8.2 (A–C) and E10.5 (D–F). Scale bars are 1000 μm.
Molecular Cell 2001 7, DOI: ( /S (01) )

8. Figure 7 Identification of a Functional ASE in Xenopus Pitx2
(A)The structures of luciferase reporter constructs and of lacZ transgene constructs containing various fragments of Xenopus Pitx2 are shown below the representation of the genomic structure of this gene. The luciferase reporter constructs were tested for their responsiveness to activin and to the constitutively active FAST-VP16 fusion protein. The lacZ transgenes were assayed for expression in E8.2 and E10.5 mouse embryos.
(B) Nucleotide sequence of the ASE region of Xenopus Pitx2, including the 0.5 kb activin-responsive AflII-SspI region. Three FAST binding sites (AATACACA, TGTGGATT, and AATAGACA) and a single Nkx2-5 binding site (CACTTTA) are shown in red and blue, respectively. The third FAST binding sequence (AATAGACA) is diverged from the consensus binding sequence, but it could bind FAST in gel shift assay (data not shown). Other sequences conversed with the mouse Pitx2 ASE are shown in green.
(C) X-gal staining of E8.2 and E10.5 mouse embryos harboring the lacZ transgene x4-P1. Left-sided staining is apparent at E8.2, whereas the transverse section of the E10.5 embryo reveals asymmetric expression in the common atrial chamber (cac) and body wall (bw) as well as bilateral staining in the posterior portion of the dorsal mesentery (dm); staining in the anterior portion of the dorsal mesentery at E10.5 was left sided (Figure 2E). Scale bars are 1000 μm.
Molecular Cell 2001 7, DOI: ( /S (01) )

9. Figure 8 A Model for Two-Step Regulation of Pitx2 Expression
The Nodal signaling pathway that includes FAST initiates the expression of Pitx2 through its ASE, thereby establishing L–R asymmetric expression at the early stage. After its activation, Pitx2 becomes accessible to other transcription factors such as Nkx2. Nkx2 is thus able to maintain the asymmetric expression of Pitx2 even in the absence of Nodal signaling. Pitx2 remains silent on the right side of embryos because Nkx2 alone is not able to activate the gene.
Molecular Cell 2001 7, DOI: ( /S (01) )