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Early patterning and specification of cardiac progenitors in gastrulating mesoderm.

Devine WP, Wythe JD, George M, Koshiba-Takeuchi K, Bruneau BG - Elife (2014)

Bottom Line: We identify Smarcd3 as a marker of early specified cardiac precursors and identify within these precursors a compartment boundary at the future junction of the left and right ventricles that arises prior to morphogenesis.Our studies define the timing and hierarchy of cardiac progenitor specification and demonstrate that the cellular and anatomical fate of mesoderm-derived cardiac cells is specified very early.These findings will be important to understand the basis of congenital heart defects and to derive cardiac regeneration strategies.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, United States.

ABSTRACT
Mammalian heart development requires precise allocation of cardiac progenitors. The existence of a multipotent progenitor for all anatomic and cellular components of the heart has been predicted but its identity and contribution to the two cardiac progenitor 'fields' has remained undefined. Here we show, using clonal genetic fate mapping, that Mesp1+ cells in gastrulating mesoderm are rapidly specified into committed cardiac precursors fated for distinct anatomic regions of the heart. We identify Smarcd3 as a marker of early specified cardiac precursors and identify within these precursors a compartment boundary at the future junction of the left and right ventricles that arises prior to morphogenesis. Our studies define the timing and hierarchy of cardiac progenitor specification and demonstrate that the cellular and anatomical fate of mesoderm-derived cardiac cells is specified very early. These findings will be important to understand the basis of congenital heart defects and to derive cardiac regeneration strategies.

No MeSH data available.


Related in: MedlinePlus

Generation of a multi-use Tbx5 allele.(A) Tbx5 targeting strategy. Top shows the wild-type (WT) allele of Tbx5: exons are indicated by roman numerals. Open boxes are untranslated regions, filled boxes are coding regions, and red box shows T-box encoding sequence. The targeting vector has a CreERT2-IRES-2X-FLAG inserted in frame with the endogenous Tbx5 coding region and a neomycin resistance cassette (neoR) flanked by frt sites (triangles) in the second intron. Flpase was used to remove the neoR cassette. (B) Southern analysis shows proper targeting of multiple independent ES cell lines. (C–N) Tbx5CreERT2 mice were crossed to RosaR26R reporter mice. Cre activity was induced at the indicated timepoints by tamoxifen injection and analyzed at E14.5. (C–H) Early induction labeled few reporter cells in the retina of double transgenic embryos, however later induction robustly labeled cells within the retina. (I–N) Early induction robustly labeled cells in the left ventricle and atria of double transgenic embryos. In addition, scattered cells are noted on the surface of the right ventricle and outflow-tract (arrows). Induction after E8.5 (L–N) continued to label the left ventricle and atria as well as the trabeculae of the right ventricle (asterisks M–N). Surface labeling on the right ventricle was no longer present after induction at E8.5. LV, left ventricle; RV, right ventricle; IVS, interventricular septum.DOI:http://dx.doi.org/10.7554/eLife.03848.021
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fig7s1: Generation of a multi-use Tbx5 allele.(A) Tbx5 targeting strategy. Top shows the wild-type (WT) allele of Tbx5: exons are indicated by roman numerals. Open boxes are untranslated regions, filled boxes are coding regions, and red box shows T-box encoding sequence. The targeting vector has a CreERT2-IRES-2X-FLAG inserted in frame with the endogenous Tbx5 coding region and a neomycin resistance cassette (neoR) flanked by frt sites (triangles) in the second intron. Flpase was used to remove the neoR cassette. (B) Southern analysis shows proper targeting of multiple independent ES cell lines. (C–N) Tbx5CreERT2 mice were crossed to RosaR26R reporter mice. Cre activity was induced at the indicated timepoints by tamoxifen injection and analyzed at E14.5. (C–H) Early induction labeled few reporter cells in the retina of double transgenic embryos, however later induction robustly labeled cells within the retina. (I–N) Early induction robustly labeled cells in the left ventricle and atria of double transgenic embryos. In addition, scattered cells are noted on the surface of the right ventricle and outflow-tract (arrows). Induction after E8.5 (L–N) continued to label the left ventricle and atria as well as the trabeculae of the right ventricle (asterisks M–N). Surface labeling on the right ventricle was no longer present after induction at E8.5. LV, left ventricle; RV, right ventricle; IVS, interventricular septum.DOI:http://dx.doi.org/10.7554/eLife.03848.021

Mentions: One class of clones identified in our Mesp1Cre-MADM clonal analysis included twin spots within the interventricular septum (IVS) between the left and right ventricles (Figure 7A–D and Figure 2). These twin spots formed a sharp boundary within the IVS, reminiscent of compartment boundaries classically defined in the Drosophila wing imaginal disc and mammalian midbrain-hindbrain boundary (Lawrence and Struhl, 1996; Dahmann and Basler, 1999). To determine if and when this boundary between the right ventricle and left ventricle is established, we performed temporally regulated genetic fate-mapping to mark early cardiac progenitors and followed their contribution later in the mature IVS. In order to mark cells that would contribute to the right ventricle, we used the Mef2c-AHF enhancer (Verzi et al., 2005) to drive expression of a fusion of the Dre recombinase (Anastassiadis et al., 2009) to the tamoxifen inducible ERT2 protein (Mef2cAHFDreERT2). We found that this enhancer element is expressed very early in the embryo, in a domain that appeared to partially overlap with that of Smarcd3 (Figure 3C,H). Previous work using a constitutive Cre recombinase expressed under the control of the Mef2cAHF enhancer suggested that endothelial and myocardial components of the outflow tract, right ventricle, and IVS are derived from this population (Verzi et al., 2005). To label the complementary population of progenitors that contribute predominantly to the left ventricle, we targeted a tamoxifen inducible Cre recombinase to the Tbx5 locus (Tbx5CreERT2) (Figure 7—figure supplement 1A–B). In the looped heart, expression of Tbx5 is restricted to the left ventricle and atria with very little expression in the right ventricle or outflow tract (Bruneau et al., 1999). Early labeling of Tbx5+ cells marked cells that were predominantly restricted to the left ventricle and atria (Figure 7E–F and 7I–L); scattered surface labeling on the right ventricle (Figure 7—figure supplement 1I–J) was also seen with early tamoxifen injection, but myocardial labeling was limited to the LV up to the junction with the RV at the IVS. Earlier observation at E8.5 following a pulse of tamoxifen at E6.5 revealed labeling in a restricted population of cells within the presumptive left ventricle (Figure 7—figure supplement 2), suggesting that the Tbx5 lineage restriction arises early and is not a consequence of later sorting out of cells. Labeling Mef2cAHF + cells at E6.5 marked a complementary population of cells that were largely restricted to the right ventricle and outflow tract (Figure 7G–H and Figure 7—figure supplement 3A). Using an intersectional reporter that responds to the combined activity of Cre and Dre we confirmed that these early Mef2cAHF + cells are also within the Smarcd3-F1 lineage (Figure 7M and Figure 7—figure supplement 3B–C). The expression of Tbx5 and the Mef2AHF enhancer appears to label complementary populations that are established prior to morphogenesis, which correspond, in part, to left and right ventricular precursors, respectively.10.7554/eLife.03848.020Figure 7.Early establishment of a boundary between the right and left ventricle at the interventricular septum.


Early patterning and specification of cardiac progenitors in gastrulating mesoderm.

Devine WP, Wythe JD, George M, Koshiba-Takeuchi K, Bruneau BG - Elife (2014)

Generation of a multi-use Tbx5 allele.(A) Tbx5 targeting strategy. Top shows the wild-type (WT) allele of Tbx5: exons are indicated by roman numerals. Open boxes are untranslated regions, filled boxes are coding regions, and red box shows T-box encoding sequence. The targeting vector has a CreERT2-IRES-2X-FLAG inserted in frame with the endogenous Tbx5 coding region and a neomycin resistance cassette (neoR) flanked by frt sites (triangles) in the second intron. Flpase was used to remove the neoR cassette. (B) Southern analysis shows proper targeting of multiple independent ES cell lines. (C–N) Tbx5CreERT2 mice were crossed to RosaR26R reporter mice. Cre activity was induced at the indicated timepoints by tamoxifen injection and analyzed at E14.5. (C–H) Early induction labeled few reporter cells in the retina of double transgenic embryos, however later induction robustly labeled cells within the retina. (I–N) Early induction robustly labeled cells in the left ventricle and atria of double transgenic embryos. In addition, scattered cells are noted on the surface of the right ventricle and outflow-tract (arrows). Induction after E8.5 (L–N) continued to label the left ventricle and atria as well as the trabeculae of the right ventricle (asterisks M–N). Surface labeling on the right ventricle was no longer present after induction at E8.5. LV, left ventricle; RV, right ventricle; IVS, interventricular septum.DOI:http://dx.doi.org/10.7554/eLife.03848.021
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4356145&req=5

fig7s1: Generation of a multi-use Tbx5 allele.(A) Tbx5 targeting strategy. Top shows the wild-type (WT) allele of Tbx5: exons are indicated by roman numerals. Open boxes are untranslated regions, filled boxes are coding regions, and red box shows T-box encoding sequence. The targeting vector has a CreERT2-IRES-2X-FLAG inserted in frame with the endogenous Tbx5 coding region and a neomycin resistance cassette (neoR) flanked by frt sites (triangles) in the second intron. Flpase was used to remove the neoR cassette. (B) Southern analysis shows proper targeting of multiple independent ES cell lines. (C–N) Tbx5CreERT2 mice were crossed to RosaR26R reporter mice. Cre activity was induced at the indicated timepoints by tamoxifen injection and analyzed at E14.5. (C–H) Early induction labeled few reporter cells in the retina of double transgenic embryos, however later induction robustly labeled cells within the retina. (I–N) Early induction robustly labeled cells in the left ventricle and atria of double transgenic embryos. In addition, scattered cells are noted on the surface of the right ventricle and outflow-tract (arrows). Induction after E8.5 (L–N) continued to label the left ventricle and atria as well as the trabeculae of the right ventricle (asterisks M–N). Surface labeling on the right ventricle was no longer present after induction at E8.5. LV, left ventricle; RV, right ventricle; IVS, interventricular septum.DOI:http://dx.doi.org/10.7554/eLife.03848.021
Mentions: One class of clones identified in our Mesp1Cre-MADM clonal analysis included twin spots within the interventricular septum (IVS) between the left and right ventricles (Figure 7A–D and Figure 2). These twin spots formed a sharp boundary within the IVS, reminiscent of compartment boundaries classically defined in the Drosophila wing imaginal disc and mammalian midbrain-hindbrain boundary (Lawrence and Struhl, 1996; Dahmann and Basler, 1999). To determine if and when this boundary between the right ventricle and left ventricle is established, we performed temporally regulated genetic fate-mapping to mark early cardiac progenitors and followed their contribution later in the mature IVS. In order to mark cells that would contribute to the right ventricle, we used the Mef2c-AHF enhancer (Verzi et al., 2005) to drive expression of a fusion of the Dre recombinase (Anastassiadis et al., 2009) to the tamoxifen inducible ERT2 protein (Mef2cAHFDreERT2). We found that this enhancer element is expressed very early in the embryo, in a domain that appeared to partially overlap with that of Smarcd3 (Figure 3C,H). Previous work using a constitutive Cre recombinase expressed under the control of the Mef2cAHF enhancer suggested that endothelial and myocardial components of the outflow tract, right ventricle, and IVS are derived from this population (Verzi et al., 2005). To label the complementary population of progenitors that contribute predominantly to the left ventricle, we targeted a tamoxifen inducible Cre recombinase to the Tbx5 locus (Tbx5CreERT2) (Figure 7—figure supplement 1A–B). In the looped heart, expression of Tbx5 is restricted to the left ventricle and atria with very little expression in the right ventricle or outflow tract (Bruneau et al., 1999). Early labeling of Tbx5+ cells marked cells that were predominantly restricted to the left ventricle and atria (Figure 7E–F and 7I–L); scattered surface labeling on the right ventricle (Figure 7—figure supplement 1I–J) was also seen with early tamoxifen injection, but myocardial labeling was limited to the LV up to the junction with the RV at the IVS. Earlier observation at E8.5 following a pulse of tamoxifen at E6.5 revealed labeling in a restricted population of cells within the presumptive left ventricle (Figure 7—figure supplement 2), suggesting that the Tbx5 lineage restriction arises early and is not a consequence of later sorting out of cells. Labeling Mef2cAHF + cells at E6.5 marked a complementary population of cells that were largely restricted to the right ventricle and outflow tract (Figure 7G–H and Figure 7—figure supplement 3A). Using an intersectional reporter that responds to the combined activity of Cre and Dre we confirmed that these early Mef2cAHF + cells are also within the Smarcd3-F1 lineage (Figure 7M and Figure 7—figure supplement 3B–C). The expression of Tbx5 and the Mef2AHF enhancer appears to label complementary populations that are established prior to morphogenesis, which correspond, in part, to left and right ventricular precursors, respectively.10.7554/eLife.03848.020Figure 7.Early establishment of a boundary between the right and left ventricle at the interventricular septum.

Bottom Line: We identify Smarcd3 as a marker of early specified cardiac precursors and identify within these precursors a compartment boundary at the future junction of the left and right ventricles that arises prior to morphogenesis.Our studies define the timing and hierarchy of cardiac progenitor specification and demonstrate that the cellular and anatomical fate of mesoderm-derived cardiac cells is specified very early.These findings will be important to understand the basis of congenital heart defects and to derive cardiac regeneration strategies.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, United States.

ABSTRACT
Mammalian heart development requires precise allocation of cardiac progenitors. The existence of a multipotent progenitor for all anatomic and cellular components of the heart has been predicted but its identity and contribution to the two cardiac progenitor 'fields' has remained undefined. Here we show, using clonal genetic fate mapping, that Mesp1+ cells in gastrulating mesoderm are rapidly specified into committed cardiac precursors fated for distinct anatomic regions of the heart. We identify Smarcd3 as a marker of early specified cardiac precursors and identify within these precursors a compartment boundary at the future junction of the left and right ventricles that arises prior to morphogenesis. Our studies define the timing and hierarchy of cardiac progenitor specification and demonstrate that the cellular and anatomical fate of mesoderm-derived cardiac cells is specified very early. These findings will be important to understand the basis of congenital heart defects and to derive cardiac regeneration strategies.

No MeSH data available.


Related in: MedlinePlus