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Keywords:

  • Pitx2c transgene;
  • P2Ztg;
  • laterality;
  • heart development

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

To aid in detection and tracking of cells targeted by the left-right (LR) pathway in the heart throughout morphogenesis, expression from a Pitx2c-lacZ transgene (P2Ztg) was analysed in detail. β-galactosidase expression from P2Ztg was robust, allowing reliable visualisation of low-level Pitx2c expression, and was virtually entirely dependent upon NODAL signalling in the heart. P2Ztg showed expression in trabecular and septal, as well as non-trabecular, myocardium, and a strong expression bias in myocardium associated with individual endocardial cushions of the atrioventricular canal and outflow tract, which are essential for cardiac septation. Expression on the ventral surface of the outflow tract evolved to a specific stripe that could be used to track the future aorta during outflow tract spiralling and remodelling. Our data show that the P2Ztg transgene is a useful resource for detection of molecular disturbances in the LR cascade, as well as morphogenetic defects associated with other cardiac congenital disorders. Developmental Dynamics, 2011. © 2010 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

The incidence of left-right (LR) malformations is around 1:5,000 to 1:10,000 in humans (Peeters and Devriendt, 2006). However, this disorder is generally under-reported, due to poor diagnosis (Maclean and Dunwoodie, 2004). Heterotaxia, in which laterality of individual tissue systems is randomised as a consequence of disturbed LR patterning during early development, is associated with complex congenital heart disease (CHD), present in 80% of cases (Maclean and Dunwoodie, 2004; Peeters and Devriendt, 2006).

The paired-like homeobox transcription factor gene, Pitx2c, is the only identified LR effector and direct target of NODAL (Shiratori et al., 2006). Pitx2 expression is retained throughout organogenesis and mediates at least some of NODAL-dependent asymmetric morphologies that play out in organ patterning in mammals (Schweickert et al., 2000). While several studies have documented the early expression pattern [8.0–9.5 days post-coitum (dpc)] of Pitx2 in the mouse, detailed expression in the heart at later developmental stages is complicated by the low level of expression of the endogenous gene. In addition, all attempts to map the fate of Pitx2c-expressing cells using transgenic or knock-in markers showed poor correlation with endogenous expression domains (Arakawa et al., 1998; Ryan et al., 1998; Gage et al., 1999; Kitamura et al., 1999; Lin et al., 1999; Lu et al., 1999; Essner et al., 2000; Hjalt et al., 2000; Schweickert et al., 2000; St Amand et al., 2000; Campione et al., 2001; Kioussi et al., 2002; Suh et al., 2002; Franco and Campione, 2003; Liu et al., 2003; Ai et al., 2006; Dong et al., 2006; Nowotschin et al., 2006; Shiratori et al., 2006).

To follow the spatial distribution of cardiac cells that receive LR information, we have analysed in detail a Pitx2c-lacZ transgenic line (17-P1 or P2Ztg) previously described (Shiratori et al., 2001). We show that the P2Ztg is a valid reporter of LR signalling in control mice and laterality mutants, and further characterise P2Ztg expression during complex morphogenetic events in cardiac development.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

P2Ztg Is a Reliable Reporter of Pitx2c Expression in the Heart

Isoform c encoded by the Pitx2 locus shares high homology with isoforms a and b. However, only Pitx2c is responsive to LR cues in mammals and the alternate isoforms have different potency in eliciting cellular or molecular responses (Essner et al., 2000; Cox et al., 2002). Therefore, specific probes for Pitx2c and Pitx2a/b isoforms and one recognizing all three isoforms (pan-Pitx2) were used to compare domains detected by X-Gal staining of embryos from the P2Ztg line (see Supp. Fig. S1, which is available online) with endogenous mRNA transcripts detected by whole mount in situ hybridisation (WISH).

Analyses were performed at 8.5–10.5 dpc (Fig. 1 and data not shown). Pitx2a/b and Pitx2c transcripts or X-Gal staining accumulated in a bilaterally symmetrical manner in the region of the branchial arches (BA) (Fig. 1A–D). While additional expression of Pitx2a/b was not detected (Fig. 1D, H), Pitx2c was abundantly transcribed in the left LPM and heart from 8.0 dpc (Fig. 1B,C, red arrowheads and data not shown). At 10.5 dpc, cardiac P2Ztg staining and Pitx2c expression were restricted to the left atrial appendage (LA), atrioventricular canal (AVC), anterior-ventral portions of the right ventricle (RV), left ventricle (LV), and outflow tract (OFT) (Fig. 1E,G). A pan-Pitx2 probe failed to reveal the existence of unexpected additional domains (Fig. 1F). Furthermore, a β-gal-specific probe revealed that P2Ztg was being transcribed at the time that X-Gal staining was detected (Fig. 1A,B). At all stages examined, X-Gal staining highlighted the same expression domains as WISH experiments with the Pitx2c probe.

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Figure 1. P2Ztg recapitulates endogenous Pitx2c expression. Whole mount X-Gal staining (A, E) or WISH (B–D; F–H) of 9.0–9.5 dpc embryos or 10.5-dpc hearts. Left-sided embryo views (A–D) and ventral heart views (E–H). β-gal (B) and Pitx2c (C) specific probes detected branchial arches (BA) bilaterally and left LPM, in a similar way to X-Gal staining from P2Ztg (A), while Pitx2a/b probe marked only BA regions (D). A pan-Pitx2 probe detects the same regions as P2Ztg (E, F). No signal was seen on the dorsal surface of the heart (not shown). Pitx2c probe (G) marked exactly the same cardiac regions as the pan-Pitx2 probe and P2Ztg (E, F), while no expression was observed for the Pitx2a/b probe in heart (H). Red arrowheads in A–D point to the heart. BA, branchial arches; LPM, lateral plate mesoderm; OFT, outflow tract; RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; AVC, atrioventricular canal.

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Nodal Mediates P2Ztg Asymmetric Expression in the Heart

Previous descriptions of Pitx2c expression in the heart have been complicated by the low levels of signal obtained in WISH experiments, while various transgenic mouse lines (Kitamura et al., 1999; Lin et al., 1999; Liu et al., 2003; Ai et al., 2006) did not fully replicate endogenous transcription. To further validate P2Ztg as a reporter of Pitx2c expression, we formally addressed whether its expression is dependent on NODAL signalling by introducing P2Ztg transgene in embryos conditionally lacking Nodal in LPM. Mice containing LoxP sites flanking the coding region of the Nodal gene (Nodalflox/flox) (Lowe et al., 2001) were intercrossed with the MesP1-Cre knock-in line (Saga et al., 1999). MesP1-Cre drives recombination in nascent mesoderm but favours the anterior mesodermal regions from which the cardiac fields derive (Furtado et al., 2008). Embryos were collected at 9–10.5 dpc and control [Nodalflox/flox; P2Ztg (n=4), Nodalflox/+;P2Ztg (n=15), andMesP1Cre/+;Nodalflox/+;P2Ztg (n=8)] or mutant (MesP1Cre/+;Nodalflox/flox;P2Ztg, n=11) embryos were analysed (Fig. 2A–D). As expected, control embryos (n=27) showed normal P2Ztg staining in the heart (Fig. 2A; Supp. Fig. S2). MesP1Cre/+;Nodalflox/flox;P2Ztg embryos displayed a wide variety of looping anomalies (C. Biben, unpublished data). Irrespective of looping shape, these mutants had a near-complete absence of P2Ztg staining in the heart. Occasional β-galactosidase (β-gal) -positive cells could be detected in the LA/sinus venosus regions (n=2) and OFT (n=2) (red asterisk in Fig. 2C,D; Supp. Fig. S2). While these rare β-gal-positive cells may have spontaneously activated the P2Ztg transgene, expression more likely reflects the partial Cre-mediated Nodal deletion in LPM (Furtado et al., 2008). These experiments strongly suggest that P2Ztg expression, as for Pitx2c, critically depends on NODAL signalling.

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Figure 2. P2Ztg expression in LR pathway mutants. A–D: Whole-mount left-sided views of control and embryos lacking Nodal in LPM, showing P2Ztg expression depends on asymmetric NODAL signalling. P2Ztg expression is consistently reduced in all mutants. Red asterisks mark a few remaining blue cells in the LA/sinus venosus compartment seen in some embryos. Red arrowhead shows intact P2Ztg staining in the branchial region. E–H: Whole-mount (E, G) or transverse sections (F, H) of control or Pitx2 mutant embryos, illustrating that P2Ztg can be used to track cells lacking Pitx2c in the heart. Black arrowheads in F, H point to transgene staining in the aorta. RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; AO, aorta; PA, pulmonary artery.

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Insertion of P2Ztg into Pitx2 conditional mutants (MesP1Cre/+; Pitx2flox/flox;P2Ztg, n=7) (Supp. Fig. S1) demonstrated that, although Pitx2 gene function was abolished, P2Ztg is still expressed, therefore allowing the tracking of Pitx2 mutant cells during heart morphogenesis (Fig. 2E–H). These cells roughly occupy similar regions as in control (MesP1Cre/+;Pitx2flox/+;P2Ztg or MesP1Cre/+;Pitx2+/+;P2Ztg, n=13) hearts, although morphogenetic defects typical of Pitx2 mutants, such as enlarged left atrial appendage and hypoplastic right ventricle, were easily visible (Fig. 2G). Furthermore, the altered shape and extent of expression domains may relate to the roles of Pitx2c in chamber remodelling (Tessari et al., 2008) and potentially in heart progenitor cell deployment during OFT morphogenesis (Lin et al., 2007).

Early P2Ztg Expression in Mesoderm-Derived Regions

To further characterize the Pitx2c expression pattern and to follow cells that received an asymmetric NODAL signal during heart morphogenesis, P2Ztg-expressing embryos and adult hearts were analysed. X-Gal staining was first detected at 8.0 dpc in left-sided LPM (Fig. 3A, black arrowheads), closely following that of Nodal expression (Lowe et al., 1996). Right-sided LPM was devoid of P2Ztg staining (Fig. 3A',B).

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Figure 3. P2Ztg expression throughout embryonic development and adulthood. Whole-mount embryonic views (A, A', B, F, L, O, P, R, and T) and transverse (C–E, G–K, Y) or longitudinal sections (M, N, O', Q, S, U–X) through the heart are shown. A–E:P2Ztg is expressed in anterior to posterior left LPM (A, black arrowheads, and B), including the cardiac crescent region (red dotted lines). Note that in right-sided view X-Gal staining on the left side can be seen due to embryo transparency (A'). At the linear heart stage, expression is retained in LPM (black dotted box in C), DM, and dorsal myocardial wall at different compartment levels (C–E). F–I: Cardiac expression encompasses IC (F and red star in G), ventral parts of cardiac chambers (F, yellow star in G), and LA/SV (G, I). Red arrowhead in D, I shows pharyngeal mesenchyme staining. J: Bilateral staining in BA. K:P2Ztg expression in posterior mesoderm. L: Dissection of atrial appendages reveals ventral P2Ztg expression on OFT (black arrowheads) and extensive LV staining. M,N: Sections show staining in interatrial septum (encircled in M) and discontinuous staining in pericardium (N, black arrowheads). O,O': Left sagittal view (O) and transverse section (O') showing X-Gal staining in blue and Nppa mRNA in purple. Red arrowheads point to double stained trabeculae. P, Q: Sharp horizontal stained boundary on ventral RV surface (red arrowheads) reflects septal staining. Q: X-Gal strongly marks compact (black arrowheads) and trabeculated (black stars) ventricular regions. R, S: P2Ztg stains AO exclusively (R), but disappears upon great vessels remodelling (T). U–Y: P2Ztg is not present in forming AVC (black stars in U) or OFT valves (dotted encircled regions in V, W). While atrial septal staining fades (U, black circle), atrial appendage (X) and ventricular (Y) stainings persist to adulthood. Red brackets in Y show blue cells along the total depth of ventricular walls; black arrowheads mark right-sided IVS staining. R, right; L, left; AL, allantois; HG, hindgut, DA, dorsal aorta; NP, neural plate; AM, amnion; HF, head folds; DM, dorsal mesocardium; OFT, outflow tract; YS, yolk sac; V, primitive ventricle; FG, foregut pocket; RA/LA, right/left atrium; AVC, atrioventricular canal; RV/LV, right/left ventricle; SV, sinus venosa; BW, body wall; MC, midgut cavity; PC, peritoneal cavity; MW, midgut wall; AO, aorta; PA, pulmonary artery; IVS, interventricular septum.

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Transverse sections showed that P2Ztg was expressed throughout left-sided mesoderm located ventral of the paraxial mesoderm, left of the hindgut, and ventral of the left branch of the dorsal aorta in the posterior region of the embryo (dashed box in Fig. 3C). P2Ztg was essentially expressed in regions of splanchnic (future gut at that level) and somatic (future body wall) mesoderm located proximal to the splanchnic/somatic junction, never reaching the embryonic midline.

Cardiac P2Ztg Expression

A frontal view at 8.0 dpc revealed that anterior LPM staining extended into the cardiac field (Fig. 3B). In sections, P2Ztg was abundantly expressed throughout dorsal left-sided myocardium, from the dorsal mesocardium (DM) into the OFT (Fig. 3C), primitive ventricle (Fig. 3D), and sinus venosus (SV) (Fig. 3E). Staining was also observed in the pharyngeal mesenchyme (Fig. 3D, I, red arrowhead). Dorsal splanchnic mesoderm and pharyngeal mesenchyme expression are considered to be part of the second heart field (Buckingham et al., 2005). Patchy staining was seen in the pericardium (arrowheads in Fig. 3N).

P2Ztg Expression in Venous Pole Regions

From 9.5 dpc onwards, a complex P2Ztg expression pattern evolved during and following the entry of second heart field progenitor cells into the heart tube (Fig. 3F–I). As the linear heart tube elongated and looped, P2Ztg expression could be detected in all cardiac chambers, except for the RA. Sections illustrated the extent of P2Ztg expression throughout the LA myocardium (Fig. 3G), left horn of SV (Fig. 3I), and DM (Fig. 3I, encircled area).

At 10.5 dpc, P2Ztg was expressed throughout the forming inter-atrial septum (Fig. 3M, encircled dashed region). In the atrial roof, P2Ztg expression extended rightward beyond the forming septum primum, into the right atrium. This staining never reached the right atrial appendage sub-domain and the positive region likely gives origin to the septum secundum, which begins as an in-folding of the myocardial roof (Anderson et al., 2003). From 16.5 dpc on, P2Ztg was down-regulated in the atrial septum (Fig. 3U, dashed encircled region), and no expression was seen in adult hearts after whole mount staining (data not shown). Endocardial staining was found in regions immediately apposed to strongly stained myocardium (Fig. 3G,H) and it is possible that such staining is an artefact generated by solution trapping or leakage of the blue X-Gal precipitation product from strongly-expressing myocardium. However, this remains unresolved.

P2Ztg Expression in the Arterial Heart Pole

P2Ztg expression was sustained at all antero-posterior levels of the dorsal linear heart tube at 8.0 dpc, although it was weaker in the OFT (Fig. 3C–E). At 10.5 dpc, a sharp boundary between ventral-positive and dorsal-negative expression domains was seen upon removal of atrial appendages (Fig. 3L, black arrowheads). Individualised aorta (AO) and pulmonary artery (PA) could be recognized as septation progressed (Fig. 3R,S). P2Ztg was then mostly off in this region, only remaining as a stripe of expressing cells extending from the RV into the right side of the AO (Fig. 3R). This stripe penetrated deep into the myocardial wall, as seen in a longitudinal section (Fig. 3S). By 16.5 dpc, this stripe was no longer evident (Fig. 3T).

P2Ztg Is Expressed in Ventricles

Ventricular myocardial staining extended from the inner curvature (IC) to the ventral surface of the OFT and RV at 9.5 dpc (Fig. 3F,G). Unlike the RV, most of the LV was devoid of X-Gal staining at this stage, with the exception of a region at the IC in continuity with the AVC (Fig. 3G, red star). Removal of atrial appendages uncovered an extensively stained region on the LV surface as chambers grow (Fig. 3L–N). Initial observations on the Pitx2 expression pattern implicated this gene as a marker of the IC (Franco and Campione, 2003), a region of less-differentiated, non-trabecular myocardium. As such, the IC does not express markers of working chamber myocardium, such as Nppa, Cx43, and Smpx. However, sections through hearts (Fig. 3N,Q) showed that X-Gal staining spanned both IC and chamber myocardium, including compact (Fig. 3Q. black arrowheads) and trabeculated regions (Fig. 3Q, black stars), showing that Pitx2 expression does not exclusively correlate with the state of cardiomyocyte or chamber differentiation. This was further confirmed by double WISH/histochemistry experiments, in which Nppa (Fig. 3O,O') and Smpx (data not shown) transcripts and P2Ztg staining were found in overlapping domains. No staining was seen on the dorsal side of the ventricles on this genetic background.

A sharp boundary of P2Ztg expression marked the interventricular (IV rpar; septal region (Fig. 3P,Q, red arrowheads). Sections revealed X-Gal staining only in the rightmost myocardial component of the septum, and not throughout the whole ventral IV ring, as suggested by Tessari and colleagues (Tessari et al., 2008). Their data may have been misinterpreted by the fact that Pitx2 is expressed in the ventral myocardial wall, continuous with the IV septum.

P2Ztg Expression Persists Postnatally

Continuous Pitx2c expression is required for correct organogenesis (Shiratori et al., 2006). Indeed, adult hearts displayed myocardial P2Ztg expression in the LA (Fig. 3X), save for some non-expressing cells at the tip of the appendage (Fig. 3Q,X). Whether those cells expressed P2Ztg initially and switched it off or have been incorporated from a negative population into the LA is unknown. In addition, P2Ztg expression extended across most of the RV and a substantial portion of LV myocardium in the ventral region (Fig. 3Y). Expressing cells spanned the whole thickness of the myocardial wall (red brackets). The external-most tissue layers in the right side of the IV septum were also stained (black arrowheads).

P2Ztg Expression Tracks Cushion Morphogenetic Events

Phenotypes induced by the absence of Pitx2 in the heart, including atrial and ventricular septal malformations, are in part associated with defects in endocardial cushions. Therefore, it is important to establish whether there is a clear relationship between Pitx2c expression and cushion morphogenesis. Sagittal sections through P2Ztg expressing embryos at 9.5 dpc (Fig. 4A–E) showed a strong correlation of X-Gal staining with myocardium surrounding the septal ridge of the OFT cushions (Fig. 4B,C).

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Figure 4. Correlation of P2Ztg expression with AVC cushions. A: Cartoon depicting embryo orientation for sagittal sections shown in B, D. B–E: Sections through OFT (B) or AVC (D) regions and respective cartoons (C, E) showing correlation between P2Ztg staining and cushion positioning at 9.5 dpc. Note blue staining on myocardial layer encasing the superior AVC cushion (D, E). F: Cartoon of transverse plane of section for G–J from a cranial view, and positioning of AVC and OFT cushions. At 10.5 dpc (G, H) and 12.5 dpc (I, J), P2Ztg partially stains myocardium underlying inferior AVC cushion in anterior-most regions (juxtaposed with LA, black arrowheads). H, head; T, tail; OFT, outflow tract; AVC, atrioventricular canal; RA/LA, right/left atrium; LV,left ventricle.

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In the AVC, X-Gal stained myocardium was seen on the ventral heart surface (Fig. 4D,E), intimately associated with the superior AVC cushion. To check whether this association was maintained throughout development, hearts were sectioned in transverse plane from 9.5 dpc to 13.5 dpc (Fig. 4F–J and data not shown). A cartoon representing cushion morphogenesis in the AVC from a cranial transverse view at 10.5 dpc is shown (Fig. 4F). During this process, swellings of extracellular matrix become obvious in both superior (blue ridge) and inferior (red ridge) regions. The superior cushion is in close contact with the OFT. Endocardial cells undergo epithelial to mesenchymal transformation (EMT) and migrate into swelled regions, which will later fuse to each other, while accessory lateral cushions will arise to aid formation of AVC valves. Completion of remodelling finalises ventricular/atrial septation (Anderson et al., 2003). In cranial sections, P2Ztg-positive myocardium surrounded the superior AVC cushion next to the base of atrial chambers (Fig. 4G). However, P2Ztg expression was seen in myocardium next to the inferior cushion, albeit only slightly (black arrowhead) at this level, in continuity with the dorsal LA expression. Deeper into the AVC (Fig. 4H), X-Gal staining exclusively enveloped the superior cushion myocardium, whilst staining was no longer visible in the inferior cushion. At 12.5 dpc (Fig. 4I,J) and 13.5 dpc (data not shown), the same correlation was seen. Conversely, the relationship between P2Ztg staining and OFT cushion positioning seen at 9.5 dpc was lost with further development. This may be due to the complex rotational events occurring during OFT remodelling (Bajolle et al. 2006).

Our analysis showed a strong correlation between the superior cushion and P2Ztg expression during cardiac development and remodelling. This correlation is observed in other developmental models. DiI labelling experiments in chick revealed a similar distribution of left and right cells in the AVC region (Campione et al., 2001). In Xenopus, right- and left-derived cells differentially labelled by red and green dextran beads demonstrated a strong correlation between the superior AVC cushion and left-derived myocardial cells (Ramsdell et al., 2005). Follow-up experiments revealed that left and right contributions were altered in heterotaxic embryos, and this phenomenon was preceded by abnormal Pitx2c expression (Ramsdell et al., 2006). In agreement with this, Smad1−/− mutant mice, which display 100% left isomerism and bilateral Nodal/P2Ztg expression, showed X-Gal staining surrounding the AVC radius (Furtado et al., 2008). Interestingly, wild type chick (Moreno-Rodriguez et al., 1997) or mouse (Webb et al., 1996) embryos normally display AVC cushion asymmetry. Whether morphological asymmetry and differential Pitx2 expression in AVC cushions are related to LR patterning remains to be addressed.

Pitx2c expression in mesenchymal cushion cells has been a contradictory topic thus far. While two groups have reported occasional X-Gal stained cells in these structures (Liu et al., 2002; Ai et al., 2006), lineage-tracing studies in mouse showed that myocardium does not transform into mesenchyme in cushions (de Lange et al., 2004), which are exclusively composed of endocardial or neural crest–derived cells. Furthermore, X-Gal staining was never seen in any of the cardiac valves (Fig. 3U–W). Both semilunar aortic (Fig. 3V) and pulmonary (Fig. 3W) valves, and the interventricular mitral and tricuspid valve leaflets (Fig. 3U, black asterisks), were devoid of β-gal, in contrast with extra-cushion myocardial tissue surrounding them. Staining did become apparent in endocardial/mesenchymal cells juxtaposed to the myocardial layer in strongly stained whole mount embryos, suggesting it was an artefact from X-gal leakage from myocardium. Further work will be necessary to clarify this issue.

P2Ztg Follows Evolving Rotational Movements in Outflow Tract Remodelling

OFT remodelling drives separation of the great vessels into PA/AO and connection with their respective ventricular chambers. Previous work by Bajolle and colleagues demonstrated that rotational events can be tracked with the y96-Myf5-nlacZ-16 transgene, which marks a subset of cells at the OFT outer curvature by 9.5 dpc (Bajolle et al., 2006). These cells move in a clockwise orientation from a cranial perspective, first staining the dorsal surface and then the right side of the OFT, culminating in a bottle-neck staining around the base of the PA by 12.5 dpc. Close observation of P2Ztg staining revealed a complementary pattern to that of y96-Myf5-nlacZ-16 (Fig. 5A–D and cartoons A'–D'). P2Ztg was initially seen in the inner curvature of OFT (Fig. 5A,A'), and a clockwise rotational evolution of this pattern led to staining covering the entire ventral (Fig. 5B,B') and part of the dorsal surfaces of this structure (Fig. 4H). This pattern was further restricted to the outermost region (right) of the OFT, which will later form the AO (Fig. 5C,C'). As septation advances, P2Ztg staining was seen in a narrow domain along the right side of the AO (Fig. 4D,D'). This domain was continuous with staining in the RV (Fig. 3R). X-Gal staining faded off following septation (Fig. 3T). In conditional Pitx2 mutant embryos, P2Ztg staining highlighted anomalous rotation of the great arteries (Fig. 2F,H). These mutants displayed 100% incidence of a side-by-side disposition of AO/PA (n=7). Our observations indicate that P2Ztg can be used to track rotational movements taking place with OFT formation and later remodelling.

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Figure 5. Evolving P2Ztg staining on OFT during great vessel remodelling. Ventral view of X-Gal stained hearts (A–D) or diagrams (A'–D') illustrating changes on dynamic P2Ztg expression pattern (blue) in the heart. Yellow arrow indicates progression of staining in a clockwise fashion. With the end of remodelling, staining is restricted to the rightmost region of the individualised aorta. AO, aorta; PA, pulmonary artery.

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Expression in Extra-Cardiac Sites

P2Ztg expression was also observed in other embryonic regions. Most sites have been previously described, and therefore will be only briefly mentioned here. At 9.5 dpc, X-Gal staining extended into mesenchymal and epithelial components of the branchial arches (Fig. 3J, black asterisks). In more posterior sections, the LPM has started to differentiate into midgut wall (MW, splanchnic component) and body wall (BW, somatic component) (Fig. 3K). Both tissues have maintained P2Ztg expression. X-Gal staining was also observed in the endodermal-derived Rathke's pouch region, precursor for the adenohypophysis (data not shown). These ectodermal/endodermal P2Ztg-expressing domains were symmetrical and independent of LPM expression.

Although P2Ztg expression matched that of the endogenous gene in sites of asymmetrical expression in the heart, it was not detected in previously reported sites such as lung buds (Kitamura et al., 1999; Liu et al., 2001) and somites (Arakawa et al., 1998; Lin et al., 1999), suggesting that either other asymmetric regulatory elements are missing in P2Ztg or the Pitx2c isoform is indeed not expressed in some or all of these sites.

Our data show that P2Ztg is a reliable tool to track LR-targeted cells that participate in heart morphogenesis, providing sensitive histochemical detection of β-gal activity, which bypasses visualisation problems due to low expression level of Pitx2c. P2Ztg is normally detected in sites where the endogenous Pitx2c gene is expressed, and it is faithfully regulated by NODAL. P2Ztg activity is seen in left-sided LPM at early steps in LR axis establishment. Previous analyses performed by our group (Furtado et al., 2008) and data presented here demonstrated that introduction of P2Ztg in LR mutant backgrounds accurately marked mispatterned regions, showing that P2Ztg is useful in the detection of left or right isomerism and most likely heterotaxia. We have also demonstrated a clear relationship between P2Ztg expression and endocardial cushion morphogenesis and OFT remodelling, showing that P2Ztg is valuable for the diagnosis of morphometric phenomena linked to LR disorders. P2Ztg can be used to track the positioning of AVC cushions and great vessels in LR mutants, therefore providing a means to re-evaluate LR-related mutants where the lack of a marker complicates phenotypic analysis.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Mouse Lines and Genotyping

Nodal conditional null (Nodalflox) (Lowe et al., 2001) and MesP1Cre (MesP1Cre) (Saga et al., 1999) mouse lines have been previously described and genotyped following original publications. These lines were maintained on a mixed C57Bl/6-129/SvJ genetic background. Pitx2 conditional null line (Pitx2flox) is described in Supp. Fig. S1. Genotyping was performed using the following set of primers: Pitx2.F 5′-ggc tat gct cac tgg ttc aga-3′; Pitx2.R1 5′-cac cga tcc agt aat gag gac-3′; Pitx2.R2 5′-tcg cta agc gct cgt cat gtt-3′. The 17-P1 reporter transgene (herein named Pitx2-lacZ or P2Ztg) was originally described by Shiratori and colleagues (Shiratori et al., 2001) (Supp. Fig. S1), maintained on a 129/SvJ background and genotyped using LacZ-specific primers (LacZF/LacZR, as below).

Whole Mount In Situ Hybridisation

Experiments were performed as previously described (Furtado et al., 2008). Probes were cloned by PCR amplification from cDNA of 8.5-dpc mouse embryos in pBluescript SK vector (Stratagene, La Jolla, CA), using specific primers: Pitx2cF 5′-gct cta gag aga gag agt gcg aga cc gaga gag-3′; Pitx2cR 5′-gcg gga tcc ctt cca gct cct gca gct gct ggc tag-3′; Pitx2a/bF 5′-gct cta gac cgg tct ctc gct ggg gtg t-3′; Pitx2a/bR 5′-gcg gat ccc tgc gac ttc agg gct gga agt atg g-3′; β-gal cDNA was cloned by PCR (LacZF 5′-cgt tca tcc ata gtt gcc tga ctc-3′; LacZR 5′-gta ata cga ctc act ata ggg ctt ccg tgt cgc cct tat tcc-3′), purified and used directly for RNA probe synthesis with T7 RNA polymerase (Roche, Nutley, NJ).

Whole Mount β-Galactosidase Staining

Embryos were fixed in 1% Paraformaldehyde/0.2% Glutaraldehyde in X-Gal buffer [5 mM EGTA, 2 mM MgCl2, 0.02% IGEPAL (NP40, Sigma, St. Louis, MO), 0.25 μM deoxychalate, 0.5 mM MgCl2, and 0.9 mM CaCl2 in PBS], washed and stained ON at 37°C in X-Gal staining solution [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6. 3H2O, and 0.5 mg/ml X-Gal substrate (Promega, Madison, WI) in X-gal buffer], washed, post-fixed in 4% PFA overnight, and kept at 4°C.

Wax Processing and Counterstaining of Embryos

X-Gal whole-mount stained embryos were dehydrated through ethanol series in H2O (25, 50, 75, and 2×100%), infused with xylene (Merck, Rahway, NJ), embedded in Paraplast (Kendall, Coral Gables, FL), and sectioned using a Leica DSC1 microtome. Slides containing embryonic sections were dewaxed, re-hydrated, and incubated in eosin solution (Sigma). Slides were again dehydrated and mounted with DePex medium (Merck).

β-Galactosidase Staining in Cryosections

Adult hearts were dissected, washed thoroughly in PBS, fixed in 1% PFA, infused with 30% sucrose, and immersed in OCT compound (Tissue Tech, Miami, FL). Embedded material was sectioned and further stained for β-gal activity followed by eosin staining and DePex mounting.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

We thank Michael Kuehn and Yumiko Saga for reagents, Mauro Costa for the careful reading of this manuscript and suggestions.

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  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_22492_sm_SuppInfoFigS1.tif188KFig. S1. Wild-type Pitx2 locus, conditional knockout and transgenic strategies. A: The Pitx2 gene locus encodes 3 different isoforms (a, b, c). All isoforms contain a C-terminal DNA-binding homeodomain, but isoform c is transcribed by an alternative promoter and has a unique 5′ exon, named 1c (Kitamura et al., 1999). Exon 1c is highly similar to exon 1 encoding the N-terminus of isoform a, suggesting that it appeared as a result of an early duplication. B: The Pitx2 conditional knockout (Pitx2floxed) targeting vector contains loxP sites flanking exon 5, including a neomycin cassette for selection. Cre-mediated deletion disrupts expression of all three Pitx2 isoforms. The intronic region between exons 4 and 5 contains a main LR regulatory element (ASE), critical for expression of Pitx2c. C: The Pitx2-lacZ transgene (P2Ztg), originally named 17-P1, spans the 5′ end of Pitx2c, as well as all coding and 3′ regions (Shiratori et al., 2001). P2Ztg constructs were microinjected in fertilized eggs and embryos were collected for validation of transgene expression by Shiratori et al. (2001). Six permanent mouse lines harbouring P2Ztg were generated, all of which showed asymmetric expression similar to Pitx2 mRNA. Two of these lines were selected for β-gal analyses, showed strong X-Gal staining and were devoid of ectopic expression. One of these lines was chosen for this work and further characterized by Southern Blot. This line had 10 integrated transgenic copies.
DVDY_22492_sm_SuppInfoFigS2.tif1885KFig. S2. P2Ztg staining in control or mutant Nodal embryos over same genetic background (C57BL6 × 129/SvJ). Embryos containing either one (A) or two copies (B) of floxed Nodal (Nodalf/+ or Nodalf/f) display normal transgene expression, while mutant embryos carrying two deleted copies of Nodal (MesP1Cre/+; Nodalf/f) show either absent heart staining (Fig. 2) or small cell populations in outflow tract and/or sinus venosum/left atrium (C).

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