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Identification of Thymosin β4 as an effector of Hand1-mediated vascular development.

Smart N, Dubé KN, Riley PR - Nat Commun (2010)

Bottom Line: We demonstrate that Hand1 binds an upstream regulatory region proximal to the promoter of Tβ4 at consensus Thing1 and E-Box sites and identify both activation and repression of Tβ4 by Hand1, through direct binding within either non-canonical or canonical E-boxes, providing new insight into gene regulation by bHLH transcription factors.Hand1-mediated activation of Tβ4 is essential for yolk sac vasculogenesis and embryonic survival, and administration of synthetic TB4 partially rescues yolk sac capillary plexus formation in Hand1- embryos.Thus, we identify an in vivo downstream target of Hand1 and reveal impaired yolk sac vasculogenesis as a primary cause of early embryonic lethality following loss of this critical bHLH factor.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Unit, UCL-Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

ABSTRACT
The bHLH transcription factor Hand1 (Heart and neural crest-derived transcript-1) has a fundamental role in cardiovascular development; however, the molecular mechanisms have not been elucidated. In this paper we identify Thymosin β4 (Tβ4/Tmsb4x), which encodes an actin monomer-binding protein implicated in cell migration and angiogenesis, as a direct target of Hand1. We demonstrate that Hand1 binds an upstream regulatory region proximal to the promoter of Tβ4 at consensus Thing1 and E-Box sites and identify both activation and repression of Tβ4 by Hand1, through direct binding within either non-canonical or canonical E-boxes, providing new insight into gene regulation by bHLH transcription factors. Hand1-mediated activation of Tβ4 is essential for yolk sac vasculogenesis and embryonic survival, and administration of synthetic TB4 partially rescues yolk sac capillary plexus formation in Hand1- embryos. Thus, we identify an in vivo downstream target of Hand1 and reveal impaired yolk sac vasculogenesis as a primary cause of early embryonic lethality following loss of this critical bHLH factor.

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TB4 rescues defective yolk sac vasculogenesis in Hand1- mutants.Whole-mount immunohistochemistry was performed on Hand1- embryos and heterozygote littermate controls at E8.5 (a–f) and E9.5 (i–r) using PECAM (a–f; i–n) and αSMA (o–r) antibodies to visualize the yolk sac capillaries and smooth muscle, respectively. A lack of appropriate capillary plexus formation (vasculogenesis) is evident in Hand1- embryos (b, e), compared with heterozygous littermates at E8.5 (a, d). Intraperitoneal (i.p.) injection of pregnant females with TB4 rescued the yolk sac vasculogenic defects and an appropriately formed vascular plexus is observed at E8.5 (c, f). ELISA revealed a significant fivefold increase in yolk sac TB4 levels 2 h after injection (6 mg kg−1, i.p.) of pregnant females (g); error bars are s.e.m., where n = mean protein concentration from six yolk sacs per treatment. ***P<0.001 (Student's t-test). TB4 concentration returns to control (uninjected or 2 h PBS) level by 48 h after injection, consistent with a mean terminal half-life of 1.8–2.1 h (human Phase IA study, RegeneRx). qRT–PCR revealed that diminished Tβ4 expression in Hand1 mutant yolk sacs was elevated to beyond the level observed in control Hand1 heterozygotes after administration of synthetic TB4 (h). Angiogenic remodelling (i, l) and smooth muscle recruitment (o, q) occurred in heterozygous embryos by E9.5 but Tβ4–rescued embryos arrested in development before undergoing these processes (k, n, p, r). Hand1- embryos are seldom recovered at E9.5. An embryo collected at this stage presented with a primitive, avascular yolk sac and was observed to undergo resorption (j, m). TB4 injection had no effect on the yolk sac vasculature of wild-type or heterozygous embryos and hence is not shown. Scale bars indicate 150 μm (a–c); 100 μm (d–f); 250 μm (i–k); 150 μm (l–n); 250 μm (o–r). AU, arbitrary units; ys, yolk sac.
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f6: TB4 rescues defective yolk sac vasculogenesis in Hand1- mutants.Whole-mount immunohistochemistry was performed on Hand1- embryos and heterozygote littermate controls at E8.5 (a–f) and E9.5 (i–r) using PECAM (a–f; i–n) and αSMA (o–r) antibodies to visualize the yolk sac capillaries and smooth muscle, respectively. A lack of appropriate capillary plexus formation (vasculogenesis) is evident in Hand1- embryos (b, e), compared with heterozygous littermates at E8.5 (a, d). Intraperitoneal (i.p.) injection of pregnant females with TB4 rescued the yolk sac vasculogenic defects and an appropriately formed vascular plexus is observed at E8.5 (c, f). ELISA revealed a significant fivefold increase in yolk sac TB4 levels 2 h after injection (6 mg kg−1, i.p.) of pregnant females (g); error bars are s.e.m., where n = mean protein concentration from six yolk sacs per treatment. ***P<0.001 (Student's t-test). TB4 concentration returns to control (uninjected or 2 h PBS) level by 48 h after injection, consistent with a mean terminal half-life of 1.8–2.1 h (human Phase IA study, RegeneRx). qRT–PCR revealed that diminished Tβ4 expression in Hand1 mutant yolk sacs was elevated to beyond the level observed in control Hand1 heterozygotes after administration of synthetic TB4 (h). Angiogenic remodelling (i, l) and smooth muscle recruitment (o, q) occurred in heterozygous embryos by E9.5 but Tβ4–rescued embryos arrested in development before undergoing these processes (k, n, p, r). Hand1- embryos are seldom recovered at E9.5. An embryo collected at this stage presented with a primitive, avascular yolk sac and was observed to undergo resorption (j, m). TB4 injection had no effect on the yolk sac vasculature of wild-type or heterozygous embryos and hence is not shown. Scale bars indicate 150 μm (a–c); 100 μm (d–f); 250 μm (i–k); 150 μm (l–n); 250 μm (o–r). AU, arbitrary units; ys, yolk sac.

Mentions: Importantly, TB4 was significantly downregulated (9.03-fold, P ≥ 0.01) in Hand1- yolk sacs to levels equivalent to background (no primary/secondary antibody-alone controls; not shown), as determined by immunostaining (Fig. 5a–f) and ImageJ analysis for quantification (Fig. 5g). Subsequent immunohistochemistry on Hand1-mutant embryos indicated a lack of vascular plexus formation in the yolk sacs of mutant embryos as compared with heterozygous littermates at E8.5 (Fig. 6a,b). Such defects in yolk sac vasculogenesis coincided, as previously reported1, with an arrest in embryonic development. In contrast, wild-type and heterozygous littermate embryos had appropriately turned and developed within a yolk sac containing a well-organized capillary plexus composed of PECAM+ endothelial cells arranged in the characteristic honeycomb pattern (Fig. 6a). Intraperitoneal injection of pregnant female mice with TB4 (6 mg kg−1) was performed to determine whether defective vasculogenesis in Hand1- yolk sacs could be rescued by TB4. Hand1- embryos treated with TB4 were recovered at E8.5 in which yolk sacs displayed an appropriately formed capillary network (PECAM immunohistochemistry, Fig. 6c–f) and in which the embryo had developed beyond the primitive arrested head fold and body axis stage of the conventional mutants. Appropriate uptake of TB4 in the yolk sac vasculature was confirmed by enzyme-linked immunosorbent assay (ELISA) on treated yolk sac tissue, which revealed a significant fivefold elevation in TB4 levels (Fig. 6g). In addition, real-time qRT–PCR revealed that reduced Tβ4 expression in Hand1 mutant yolk sacs was elevated beyond the level observed in control Hand1 heterozygotes after administration of synthetic TB4 (Fig. 6h); this not only further confirmed appropriate localization of TB4 treatment to the developing yolk sac in utero, but also suggested an autoregulatory effect of TB4 on its own gene expression. In two independent Hand1- lines (conventional targeted Hand1 allele and a tTA knock-in to the Hand1 locus), the mutant phenotype at E8.5 is virtually 100% penetrant115, as confirmed in all vehicle-treated mutants examined in this study, suggesting that TB4-rescued mutants do not simply represent Hand1 mutants presenting with a less-severe phenotype. Therefore, we conclude that TB4 is sufficient to rescue vasculogenic defects in the embryonic yolk sac that arise because of an absence of Hand1. Conventional Hand1 mutants were rarely observed at E9.5 (n = 1 from 5 litters/49 embryos, expected number=12.5; 25%) and those that could be isolated displayed gross phenotypical defects compared with heterozygous littermates (Fig. 6i), leading to resorption (Fig. 6j); however, TB4-rescued Hand1 mutants were recovered, but had evidently arrested in development by this stage (Fig. 6k). At E9.5, TB4-rescued Hand1- embryos were recovered with 16–18 somite pairs, compared with 17–23 in wild-type/heterozygous littermates (based on n = 6 litters), representing a clear progression in development beyond the 8–12 somite pairs observed at E8.5. The E9.5 'rescued' embryos lacked large, blood-filled, vitelline vessels and the accompanying highly organized meshwork of secondary smaller vessels apparent in wild-type yolk sac at the equivalent stage (Fig. 6l–r). This suggests that Tβ4 rescued early yolk sac vasculogenesis at E8.5 but was insufficient to rescue angiogenic remodelling of the primary vascular plexus at E9.5 to ensure continued embryonic growth and survival.


Identification of Thymosin β4 as an effector of Hand1-mediated vascular development.

Smart N, Dubé KN, Riley PR - Nat Commun (2010)

TB4 rescues defective yolk sac vasculogenesis in Hand1- mutants.Whole-mount immunohistochemistry was performed on Hand1- embryos and heterozygote littermate controls at E8.5 (a–f) and E9.5 (i–r) using PECAM (a–f; i–n) and αSMA (o–r) antibodies to visualize the yolk sac capillaries and smooth muscle, respectively. A lack of appropriate capillary plexus formation (vasculogenesis) is evident in Hand1- embryos (b, e), compared with heterozygous littermates at E8.5 (a, d). Intraperitoneal (i.p.) injection of pregnant females with TB4 rescued the yolk sac vasculogenic defects and an appropriately formed vascular plexus is observed at E8.5 (c, f). ELISA revealed a significant fivefold increase in yolk sac TB4 levels 2 h after injection (6 mg kg−1, i.p.) of pregnant females (g); error bars are s.e.m., where n = mean protein concentration from six yolk sacs per treatment. ***P<0.001 (Student's t-test). TB4 concentration returns to control (uninjected or 2 h PBS) level by 48 h after injection, consistent with a mean terminal half-life of 1.8–2.1 h (human Phase IA study, RegeneRx). qRT–PCR revealed that diminished Tβ4 expression in Hand1 mutant yolk sacs was elevated to beyond the level observed in control Hand1 heterozygotes after administration of synthetic TB4 (h). Angiogenic remodelling (i, l) and smooth muscle recruitment (o, q) occurred in heterozygous embryos by E9.5 but Tβ4–rescued embryos arrested in development before undergoing these processes (k, n, p, r). Hand1- embryos are seldom recovered at E9.5. An embryo collected at this stage presented with a primitive, avascular yolk sac and was observed to undergo resorption (j, m). TB4 injection had no effect on the yolk sac vasculature of wild-type or heterozygous embryos and hence is not shown. Scale bars indicate 150 μm (a–c); 100 μm (d–f); 250 μm (i–k); 150 μm (l–n); 250 μm (o–r). AU, arbitrary units; ys, yolk sac.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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f6: TB4 rescues defective yolk sac vasculogenesis in Hand1- mutants.Whole-mount immunohistochemistry was performed on Hand1- embryos and heterozygote littermate controls at E8.5 (a–f) and E9.5 (i–r) using PECAM (a–f; i–n) and αSMA (o–r) antibodies to visualize the yolk sac capillaries and smooth muscle, respectively. A lack of appropriate capillary plexus formation (vasculogenesis) is evident in Hand1- embryos (b, e), compared with heterozygous littermates at E8.5 (a, d). Intraperitoneal (i.p.) injection of pregnant females with TB4 rescued the yolk sac vasculogenic defects and an appropriately formed vascular plexus is observed at E8.5 (c, f). ELISA revealed a significant fivefold increase in yolk sac TB4 levels 2 h after injection (6 mg kg−1, i.p.) of pregnant females (g); error bars are s.e.m., where n = mean protein concentration from six yolk sacs per treatment. ***P<0.001 (Student's t-test). TB4 concentration returns to control (uninjected or 2 h PBS) level by 48 h after injection, consistent with a mean terminal half-life of 1.8–2.1 h (human Phase IA study, RegeneRx). qRT–PCR revealed that diminished Tβ4 expression in Hand1 mutant yolk sacs was elevated to beyond the level observed in control Hand1 heterozygotes after administration of synthetic TB4 (h). Angiogenic remodelling (i, l) and smooth muscle recruitment (o, q) occurred in heterozygous embryos by E9.5 but Tβ4–rescued embryos arrested in development before undergoing these processes (k, n, p, r). Hand1- embryos are seldom recovered at E9.5. An embryo collected at this stage presented with a primitive, avascular yolk sac and was observed to undergo resorption (j, m). TB4 injection had no effect on the yolk sac vasculature of wild-type or heterozygous embryos and hence is not shown. Scale bars indicate 150 μm (a–c); 100 μm (d–f); 250 μm (i–k); 150 μm (l–n); 250 μm (o–r). AU, arbitrary units; ys, yolk sac.
Mentions: Importantly, TB4 was significantly downregulated (9.03-fold, P ≥ 0.01) in Hand1- yolk sacs to levels equivalent to background (no primary/secondary antibody-alone controls; not shown), as determined by immunostaining (Fig. 5a–f) and ImageJ analysis for quantification (Fig. 5g). Subsequent immunohistochemistry on Hand1-mutant embryos indicated a lack of vascular plexus formation in the yolk sacs of mutant embryos as compared with heterozygous littermates at E8.5 (Fig. 6a,b). Such defects in yolk sac vasculogenesis coincided, as previously reported1, with an arrest in embryonic development. In contrast, wild-type and heterozygous littermate embryos had appropriately turned and developed within a yolk sac containing a well-organized capillary plexus composed of PECAM+ endothelial cells arranged in the characteristic honeycomb pattern (Fig. 6a). Intraperitoneal injection of pregnant female mice with TB4 (6 mg kg−1) was performed to determine whether defective vasculogenesis in Hand1- yolk sacs could be rescued by TB4. Hand1- embryos treated with TB4 were recovered at E8.5 in which yolk sacs displayed an appropriately formed capillary network (PECAM immunohistochemistry, Fig. 6c–f) and in which the embryo had developed beyond the primitive arrested head fold and body axis stage of the conventional mutants. Appropriate uptake of TB4 in the yolk sac vasculature was confirmed by enzyme-linked immunosorbent assay (ELISA) on treated yolk sac tissue, which revealed a significant fivefold elevation in TB4 levels (Fig. 6g). In addition, real-time qRT–PCR revealed that reduced Tβ4 expression in Hand1 mutant yolk sacs was elevated beyond the level observed in control Hand1 heterozygotes after administration of synthetic TB4 (Fig. 6h); this not only further confirmed appropriate localization of TB4 treatment to the developing yolk sac in utero, but also suggested an autoregulatory effect of TB4 on its own gene expression. In two independent Hand1- lines (conventional targeted Hand1 allele and a tTA knock-in to the Hand1 locus), the mutant phenotype at E8.5 is virtually 100% penetrant115, as confirmed in all vehicle-treated mutants examined in this study, suggesting that TB4-rescued mutants do not simply represent Hand1 mutants presenting with a less-severe phenotype. Therefore, we conclude that TB4 is sufficient to rescue vasculogenic defects in the embryonic yolk sac that arise because of an absence of Hand1. Conventional Hand1 mutants were rarely observed at E9.5 (n = 1 from 5 litters/49 embryos, expected number=12.5; 25%) and those that could be isolated displayed gross phenotypical defects compared with heterozygous littermates (Fig. 6i), leading to resorption (Fig. 6j); however, TB4-rescued Hand1 mutants were recovered, but had evidently arrested in development by this stage (Fig. 6k). At E9.5, TB4-rescued Hand1- embryos were recovered with 16–18 somite pairs, compared with 17–23 in wild-type/heterozygous littermates (based on n = 6 litters), representing a clear progression in development beyond the 8–12 somite pairs observed at E8.5. The E9.5 'rescued' embryos lacked large, blood-filled, vitelline vessels and the accompanying highly organized meshwork of secondary smaller vessels apparent in wild-type yolk sac at the equivalent stage (Fig. 6l–r). This suggests that Tβ4 rescued early yolk sac vasculogenesis at E8.5 but was insufficient to rescue angiogenic remodelling of the primary vascular plexus at E9.5 to ensure continued embryonic growth and survival.

Bottom Line: We demonstrate that Hand1 binds an upstream regulatory region proximal to the promoter of Tβ4 at consensus Thing1 and E-Box sites and identify both activation and repression of Tβ4 by Hand1, through direct binding within either non-canonical or canonical E-boxes, providing new insight into gene regulation by bHLH transcription factors.Hand1-mediated activation of Tβ4 is essential for yolk sac vasculogenesis and embryonic survival, and administration of synthetic TB4 partially rescues yolk sac capillary plexus formation in Hand1- embryos.Thus, we identify an in vivo downstream target of Hand1 and reveal impaired yolk sac vasculogenesis as a primary cause of early embryonic lethality following loss of this critical bHLH factor.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Unit, UCL-Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

ABSTRACT
The bHLH transcription factor Hand1 (Heart and neural crest-derived transcript-1) has a fundamental role in cardiovascular development; however, the molecular mechanisms have not been elucidated. In this paper we identify Thymosin β4 (Tβ4/Tmsb4x), which encodes an actin monomer-binding protein implicated in cell migration and angiogenesis, as a direct target of Hand1. We demonstrate that Hand1 binds an upstream regulatory region proximal to the promoter of Tβ4 at consensus Thing1 and E-Box sites and identify both activation and repression of Tβ4 by Hand1, through direct binding within either non-canonical or canonical E-boxes, providing new insight into gene regulation by bHLH transcription factors. Hand1-mediated activation of Tβ4 is essential for yolk sac vasculogenesis and embryonic survival, and administration of synthetic TB4 partially rescues yolk sac capillary plexus formation in Hand1- embryos. Thus, we identify an in vivo downstream target of Hand1 and reveal impaired yolk sac vasculogenesis as a primary cause of early embryonic lethality following loss of this critical bHLH factor.

Show MeSH
Related in: MedlinePlus