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First published online July 11, 2006
doi: 10.1242/10.1242/dev.02470

Development 133, 3015-3025 (2006)
Published by The Company of Biologists 2006
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Conserved regulation and role of Pitx2 in situs-specific morphogenesis of visceral organs

Hidetaka Shiratori1,*, Kenta Yashiro1, Michael M. Shen2 and Hiroshi Hamada1,*

1 Developmental Genetics Group, Graduate School for Frontier Biosciences, Osaka University, and CREST, Japan Science and Technology Corporation (JST), 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan.
2 Center for Advanced Biotechnology and Medicine and Department of Pediatrics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.


Figure 1
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  		Fig. 1. Conservation of an asymmetric enhancer (ASE) of Pitx2 among vertebrates. Structures of the Pitx2 ASE from various vertebrates (located in the last intron of Pitx2) are shown on the left. Red and blue circles represent Foxh1- and Nkx2-binding sites, respectively, with the arrows indicating their orientation. The enhancer activity of each ASE in transgenic mouse assays at E8.2 and 10.5 is summarized on the right. The ASE of zebrafish Pitx2 possesses activity only in the anterior portion of left LPM (asterisk). ND, not determined.

 

Figure 2
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  		Fig. 2. Activity of the Pitx2 ASE from various vertebrates. Reporter constructs (hsp68-lacZ) driven by the Pitx2 ASE from various vertebrates were injected into fertilized mouse eggs, and the resulting embryos were stained with X-gal. (A-E) Frontal and lateral views of transgenic mouse embryos at E8.2. All constructs examined gave rise to asymmetric X-gal staining in left LPM, although the zebrafish ASE was active only in the anterior portion of left LPM. (F-Y) Transverse sections of embryos at E10.5. Activities in the common atrial chamber (F-J), foregut (lung bud) (K-O), septum transversum (future liver) (P-T), and gut dorsal mesentery (U-Y) are shown. Red arrows indicate the domains in which the ASE was specifically active. Encircled + or - signs indicate whether the ASE was active or inactive, respectively. The ASE from mouse, human or chicken Pitx2 showed left-sided activity in the indicated organs. The frog ASE was active in the common atrial chamber and gut dorsal mesentery, but inactive in the foregut and septum transversum. The zebrafish ASE was inactive in all organs. The number of the embryos examined and variations in expression patterns are shown in Table S1 of the supplementary material. Scale bars: 200 µm. A, anterior; L, left; P, posterior; R, right.

 

Figure 3
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  		Fig. 3. Prolongation of the transient enhancer activity of the ASE of Lefty2 by addition of Nkx2-binding sequences. (A,B) Structures of two lacZ transgenes are shown at the top. Red circles and blue ovals indicate Foxh1- and Nkx2-binding sequences, respectively. The partially blue circles and boxes indicate the hsp68 promoter and lacZ, respectively. The construct in A includes the Lefty2 ASE, which contains two Foxh1-binding sequences. The construct in B contains the Lefty2 ASE plus seven tandem repeats of the Nkx2-binding sequence derived from the Pitx2 ASE. X-gal staining patterns of E8.2 mouse embryos harboring each transgene are shown below. Both lacZ constructs gave rise to typical asymmetric staining in left LPM. (C-H) Transverse sections of X-gal-stained transgenic embryos at E10.5. Expression of lacZ is shown for the common atrial chamber (C,D), foregut (E,F), and gut dorsal mesentery (G,H). Black arrows indicate the domains where asymmetric X-gal staining was detected. Encircled + or - signs indicate whether the enhancer was active or inactive, respectively. The Lefty2 ASE construct did not give rise to asymmetric staining (C,E,G), whereas the N7-Lefty2 ASE construct did so in the common cardinal vein, lung bud and gut dorsal mesentery (D,F,H). Scale bars: 200 µm.

 

Figure 4
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  		Fig. 4. Generation of mice that lack the ASE of Pitx2. (A) Schematic representation of the mouse wild-type Pitx2 allele, a targeting vector designed to delete the 0.6 kb region of Pitx2 that contains the ASE (red oval), as well as the targeted allele before and after Cre-mediated recombination. Pitx2 contains six exons (black boxes) and two promoters, with transcription beginning at exon 1 or exon 1c. (B) Southern blot analysis of Nsi-digested DNA from ES cells of the indicated Pitx2 genotypes before Cre-mediated recombination. The probe used for hybridization is shown by the blue bar on the top. The wild-type and mutant alleles give rise to hybridizing fragments of >23 and 11 kb, respectively. Green triangles, loxP sequences; neo, neomycin resistance gene under the control of the phosphoglycerate kinase gene promoter; DT, diphtheria toxin gene.

 

Figure 5
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  		Fig. 5. Loss of most LR asymmetric expression domains of Pitx2 in Pitx2{Delta}ASE/{Delta}ASE embryos. Expression of Pitx2 was examined in wild-type (A,D,G,J,M,P), Pitx2{Delta}ASE/{Delta}ASE (B,E,H,K,N,Q) or cryptic-/- (C,F,I,L,O,R) embryos at E8.2 (A-C), E9.0 (D-F) or E9.5 (G-R) by whole-mount in-situ hybridization. The whole-mount embryos shown in G-I were sectioned transversely at the indicated planes and the sections are shown in J-R. Expression of Pitx2 in left LPM at E8.2 was lost in Pitx2{Delta}ASE/{Delta}ASE and cryptic-/- embryos. Asymmetric Pitx2 expression was lost in Pitx2{Delta}ASE/{Delta}ASE embryos at E9.0, with the exception of a reduced level of expression remaining in the common cardinal vein and vitelline vein (green arrow in E). Asymmetric Pitx2 expression was completely absent from cryptic-/- embryos at E9.0. Asymmetric Pitx2 expression remained apparent at a reduced level in the common cardinal vein and vitelline vein of Pitx2{Delta}ASE/{Delta}ASE embryos at E9.5 (green arrows in H, N and Q) but was absent from cryptic-/- embryos at this time. Scale bars: 200 µm. cac, common atrial chamber; ccv, common cardinal vein; dm, gut dorsal mesentery; lb, lung bud; st, septum transversum; vv, vitelline vein.

 

Figure 6
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  		Fig. 6. LR defects in Pitx2{Delta}ASE/{Delta}ASE mice. Visceral organs and heart sections of wild-type (+/+) and Pitx2{Delta}ASE/{Delta}ASE neonates are shown in A-H,M-V. (A,B) The left and right lungs of wild-type mice have one and four lobes (indicated in yellow), respectively, whereas the lungs of all Pitx2{Delta}ASE/{Delta}ASE mice had four lobes on each side. (C,D) The relative positions of the aorta (ao, blue asterisk) and pulmonary artery (pa, yellow asterisk) are reversed in Pitx2{Delta}ASE/{Delta}ASE mice. The position of the heart apex is toward the left in wild-type mice but was reversed (toward the right) in 28% of Pitx2{Delta}ASE/{Delta}ASE mice. The inferior vena cava is present only on the right side of the thorax in wild-type mice (C, blue arrowhead) but was bilateral in 28% of Pitx2{Delta}ASE/{Delta}ASE mice (D, two arrowheads). (E,F) External morphology of the atrium showing right isomerism in Pitx2{Delta}ASE/{Delta}ASE mice. In wild-type mice, the left atrium has a narrow junction to the venous component (green line in E), whereas the right atrium has a wide junction. In Pitx2{Delta}ASE/{Delta}ASE mice, both the left (green line in F) and right atria exhibited a morphology similar to that of the right atrium of the wild type. (G,H) Frontal sections of the heart. Atrial septal defect was apparent in the mutant heart. (I-L) AV cushions of the heart at E12.5 are shown by scanning electron microscopy (SEM) (I,J) and by sagittal sectioning of HE-stained hearts (K,L). SAVC and IAVC are fused with each other in the wild-type heart, but they remain separated in the Pitx2{Delta}ASE/{Delta}ASE embryos. The yellow arrowheads in K denote the boundary between SAVC and IAVC. The dotted area in L indicates a portion of SAVC that is missing in the Pitx2{Delta}ASE/{Delta}ASE embryos. (M,N) The azygos vein and thoracic aorta are located on the left side of the thorax in wild-type mice, whereas they were present on the right side in some Pitx2{Delta}ASE/{Delta}ASE mice. (O,P) The portal vein passes dorsally to the duodenum and is not visible from the ventral side in wild-type mice, but it was on the ventral side of the duodenum in some Pitx2{Delta}ASE/{Delta}ASE mice (black arrow in P). (Q-T) Abnormal rotation of the gut, including aberrant looping of the duodenum (S) or the lack of a cross of the duodenum and colon (T), was apparent in Pitx2{Delta}ASE/{Delta}ASE mice at a high frequency. Blue arrows indicate positioning of the gut. The pancreas, which is located on the left side of the duodenum and is located behind the ileum in a ventral view in wild-type mice (Q), was positioned on the ventral or right side of the duodenum in Pitx2{Delta}ASE/{Delta}ASE mice (R). (U,V) In the abdomen of wild-type mice, the aorta and vena cava are located on the left and right sides of the midline, respectively; their relative positions were reversed in 25% of Pitx2{Delta}ASE/{Delta}ASE mice. Scale bars: 200 µm. ao, aorta; az, azygos vein; co, colon; du, duodenum; IAVC, inferior atrioventricular cushion; la, left atrium; lv, left ventricle; pc, pancreas; ra, right atrium; rv, right ventricle; SAVC, superior atrioventricular cushion; ta, thoracic aorta; vc, vena cava.

 

Figure 7
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  		Fig. 7. Expression patterns of genes related to Nodal signaling. (A,D,G,J,M,P,S) Expression of cryptic was examined in wild-type mouse embryos by whole-mount in-situ hybridization. Expression was bilateral in LPM at E8.2 (red arrowheads in A) but was not detected at E9.0 (D,G,J) or E9.5 (M,P,S). (B,E,H,K,N,Q,T) Nodal expression was examined by X-gal staining of NodallacZ/+ embryos. Staining was apparent in left LPM at E8.2 (B), was detected at a low level in the heart region containing the common cardinal vein and vitelline vein at E9.0 (E,H,K) and was not observed at E9.5 (N,Q,T). (C,F,I,L,O,R,U) Foxh1 expression was examined by X-gal staining of embryos expressing a Foxh1-lacZ transgene. Staining was apparent in LPM and heart primordial cells at E8.2 (C) and was evident in the heart, but not in the common cardinal vein and vitelline vein, at E9.0 (F,I,L) and E9.5 (O,R,U). Transverse sections were prepared after whole-mount in-situ hybridization (G,J,P,S) or X-gal staining (H,I,K,L,Q,R,T,U). Black and yellow arrowheads in D-U indicate the region that corresponds to the common cardinal vein and vitelline vein and in which asymmetric Pitx2 expression persists in Pitx2{Delta}ASE/{Delta}ASE embryos. Scale bars: 200 µm.

 

Figure 8
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  		Fig. 8. Regulation of Pitx2 expression by negative feedback in the ventricle and AV canal of the heart. (A-H) Expression of the Pitx2 17-lacZ transgene in the heart of wild-type (A-D) or Pitx2{Delta}ASE/{Delta}ASE (E-H) embryos at E9.5. (I-P) Expression of the Pitx2-Cre transgene in wild-type (I-L) or Pitx2{Delta}ASE/{Delta}ASE (M-P) embryos at E9.5 as monitored with the CAG-CAT-lacZ transgene. A left view of the heart is shown in the left-most panels. Red dotted circles indicate a large portion of the ventricle and AV canal in which X-gal staining is absent in A but present in E, I and M. The transverse sections of the heart at the planes indicated by the yellow and red arrowheads are shown in B,F,J,N and C,G,K,O, respectively. The right-most panels show sagittal sections at the AV canal. Their planes are indicated by the red vertical lines in A,E,I,M. The boundaries between SAVC and IAVC in D,H,L,P are indicated by the orange dotted lines. It should be noted that X-gal staining is apparent only at the myocardium adjacent to the SAVC in D, while it is also apparent in the myocardium adjacent to the IAVC (H,L,P). The domain that is subject to the negative feedback regulation is indicated by the yellow bar in D,H,L,P. Scale bars: 200 µm. la, left atrium; lv, ventricle of the heart.

 

Figure 9
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  		Fig. 9. Generation of transgenic mice that express Pitx2c under the control of the ASE. (A) Construction of two transgenes. Expression of the Pitx2c cDNA and lacZ is driven either by the wild-type ASE or a mutant version (NmASE) that lacks the Nkx2-binding site. Permanent transgenic mouse lines were established for the former (Tg39, Tg55) and latter (Tg50) transgenes. Red and blue ovals represent Foxh1-binding sites and the Nkx2-binding site, respectively. (B-M) Expression of the transgenes in each transgenic line was monitored by X-gal staining. The transgenes in each line (Tg39, Tg55, Tg50) were expressed at similar levels in left LPM at E8.2 (B-D). The transgene of line Tg39 was asymmetrically expressed in the truncus arteriosus at a high level (E), in gut dorsal mesentery at a low level (K) and not at all in the lung bud (H) at E10.5. Asymmetric expression of the transgene in line Tg55 was apparent at a high level in gut dorsal mesentery (L), at a low level in the truncus arteriosus (F) and not at all in the lung bud (I). Asymmetric expression of the transgene in line Tg50 was not detected in any organ examined (G,J,M). Frontal and lateral views of embryos are shown in B-D, and transverse sections are shown in E-M. Scale bars: 200 µm.

 

Figure 10
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  		Fig. 10. Abilities of the Tg39, Tg55 or Tg50 transgenes to rescue the LR defects of Pitx2{Delta}ASE/{Delta}ASE mice. Phenotypes of Pitx2{Delta}ASE/{Delta}ASE neonates expressing the transgenes of lines Tg39 (A,D,G), Tg55 (B,E,H) or Tg50 (C,F,I) are shown. The transgene of line Tg39 rescued the great artery defect (A), whereas that of line Tg55 rescued the abnormal rotation of the gut (H) apparent in Pitx2{Delta}ASE/{Delta}ASE mice. The transgene of line Tg50 was unable to rescue any of the defects examined (C,F,I). Right isomerism of the lungs was not rescued by any of the transgenes (D-F); the number of lobes is indicated in yellow. Blue arrows in G-I indicate the location of the duodenum. Encircled + or - signs indicate whether LR defects were rescued or not, respectively. ao, aorta (blue asterisk); pa, pulmonary artery (yellow asterisk); pc, pancreas.

 

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© The Company of Biologists Ltd 2006