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A novel TGFβ modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback.

Gu W, Monteiro R, Zuo J, Simões FC, Martella A, Andrieu-Soler C, Grosveld F, Sauka-Spengler T, Patient R - PLoS Biol. (2015)

Bottom Line: Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback.In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP.Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

ABSTRACT
As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

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Knockdown of ldb2a enhances Nodal/BMP signal transduction in zebrafish embryos.(A) At the onset of gastrulation, ldb2a expression is more noticeable in the yolk syncytial layer (YSL) (white arrowheads), which is an important source of Nodal signals (purple arrows). (B) At the shield stage, western blot showed increased phosphorylation of R-Smad2 in ldb2a morphants, while the level of overall Smad2/3 remained the same. β-actin was used as a loading control. (C) The activity of a TGFβ reporter SBE-luciferase was up-regulated in shield stage ldb2a morphants. (D) Phosphorylation of R-Smad1/5/8 was up-regulated in ldb2a morphants at the tailbud stage. β-tubulin was the loading control. (E) The activity of a BMP reporter Id1-BRE2-luciferase was increased in tailbud stage ldb2a morphants. (C and E) each displays a single representative experiment of three biological replicates, with the error bars corresponding to two technical replicates. For each biological replicate, 50 embryos were lysed and analysed. (F–I’) Expression of Nodal ligands, cyc and sqt, was increased in shield stage ldb2a morphants. (J–K’) Anterior and posterior expression of bmp4 was up-regulated at the tailbud stage. (F–G’): seven independent experiments, with the total number of analysed embryos shown on the top-right corner of each panel; (H–K’): two independent experiments. The wildtype control refers to uninjected embryos that are stage matched.
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pbio.1002051.g002: Knockdown of ldb2a enhances Nodal/BMP signal transduction in zebrafish embryos.(A) At the onset of gastrulation, ldb2a expression is more noticeable in the yolk syncytial layer (YSL) (white arrowheads), which is an important source of Nodal signals (purple arrows). (B) At the shield stage, western blot showed increased phosphorylation of R-Smad2 in ldb2a morphants, while the level of overall Smad2/3 remained the same. β-actin was used as a loading control. (C) The activity of a TGFβ reporter SBE-luciferase was up-regulated in shield stage ldb2a morphants. (D) Phosphorylation of R-Smad1/5/8 was up-regulated in ldb2a morphants at the tailbud stage. β-tubulin was the loading control. (E) The activity of a BMP reporter Id1-BRE2-luciferase was increased in tailbud stage ldb2a morphants. (C and E) each displays a single representative experiment of three biological replicates, with the error bars corresponding to two technical replicates. For each biological replicate, 50 embryos were lysed and analysed. (F–I’) Expression of Nodal ligands, cyc and sqt, was increased in shield stage ldb2a morphants. (J–K’) Anterior and posterior expression of bmp4 was up-regulated at the tailbud stage. (F–G’): seven independent experiments, with the total number of analysed embryos shown on the top-right corner of each panel; (H–K’): two independent experiments. The wildtype control refers to uninjected embryos that are stage matched.

Mentions: To analyse the role of Ldbs in TGFβ signalling in vivo, we first monitored their expression during early embryonic development when TGFβ family members are known to be critical. Throughout early zebrafish development, ldb2a shows greater specificity than the ubiquitous ldb1a, ldb1b, or ldb2b (S2 Fig. and data retrieved from the Zebrafish Information Network (ZFIN) [30]). At 15 hours post fertilisation (hpf), ldb2a is present in the notochord and the lateral mesoderm, which gives rise to haematopoietic, endothelial, and pronephric derivatives (S2A Fig.). At 26 hpf, ldb2a expression continues in and around the blood vessels (S2B Fig.). Maternal/zygotic ldb2a is expressed ubiquitously throughout cleavage and blastula stage (0–4.7 hpf) embryos (S2C–S2F Fig.), but immediately before and during gastrulation (4.7–10 hpf), ldb2a becomes more specific in the yolk syncytial layer (YSL) (Figs. 2A and S2F, white arrowheads), an important source of Nodal signalling crucial for the specification of gastrula germ layers. This suggests a possible role for Ldb2a in signalling by this TGFβ superfamily member, we therefore initially focussed our studies on the function of Ldb2a in Nodal signalling during gastrula embryonic development.


A novel TGFβ modulator that uncouples R-Smad/I-Smad-mediated negative feedback from R-Smad/ligand-driven positive feedback.

Gu W, Monteiro R, Zuo J, Simões FC, Martella A, Andrieu-Soler C, Grosveld F, Sauka-Spengler T, Patient R - PLoS Biol. (2015)

Knockdown of ldb2a enhances Nodal/BMP signal transduction in zebrafish embryos.(A) At the onset of gastrulation, ldb2a expression is more noticeable in the yolk syncytial layer (YSL) (white arrowheads), which is an important source of Nodal signals (purple arrows). (B) At the shield stage, western blot showed increased phosphorylation of R-Smad2 in ldb2a morphants, while the level of overall Smad2/3 remained the same. β-actin was used as a loading control. (C) The activity of a TGFβ reporter SBE-luciferase was up-regulated in shield stage ldb2a morphants. (D) Phosphorylation of R-Smad1/5/8 was up-regulated in ldb2a morphants at the tailbud stage. β-tubulin was the loading control. (E) The activity of a BMP reporter Id1-BRE2-luciferase was increased in tailbud stage ldb2a morphants. (C and E) each displays a single representative experiment of three biological replicates, with the error bars corresponding to two technical replicates. For each biological replicate, 50 embryos were lysed and analysed. (F–I’) Expression of Nodal ligands, cyc and sqt, was increased in shield stage ldb2a morphants. (J–K’) Anterior and posterior expression of bmp4 was up-regulated at the tailbud stage. (F–G’): seven independent experiments, with the total number of analysed embryos shown on the top-right corner of each panel; (H–K’): two independent experiments. The wildtype control refers to uninjected embryos that are stage matched.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4321984&req=5

pbio.1002051.g002: Knockdown of ldb2a enhances Nodal/BMP signal transduction in zebrafish embryos.(A) At the onset of gastrulation, ldb2a expression is more noticeable in the yolk syncytial layer (YSL) (white arrowheads), which is an important source of Nodal signals (purple arrows). (B) At the shield stage, western blot showed increased phosphorylation of R-Smad2 in ldb2a morphants, while the level of overall Smad2/3 remained the same. β-actin was used as a loading control. (C) The activity of a TGFβ reporter SBE-luciferase was up-regulated in shield stage ldb2a morphants. (D) Phosphorylation of R-Smad1/5/8 was up-regulated in ldb2a morphants at the tailbud stage. β-tubulin was the loading control. (E) The activity of a BMP reporter Id1-BRE2-luciferase was increased in tailbud stage ldb2a morphants. (C and E) each displays a single representative experiment of three biological replicates, with the error bars corresponding to two technical replicates. For each biological replicate, 50 embryos were lysed and analysed. (F–I’) Expression of Nodal ligands, cyc and sqt, was increased in shield stage ldb2a morphants. (J–K’) Anterior and posterior expression of bmp4 was up-regulated at the tailbud stage. (F–G’): seven independent experiments, with the total number of analysed embryos shown on the top-right corner of each panel; (H–K’): two independent experiments. The wildtype control refers to uninjected embryos that are stage matched.
Mentions: To analyse the role of Ldbs in TGFβ signalling in vivo, we first monitored their expression during early embryonic development when TGFβ family members are known to be critical. Throughout early zebrafish development, ldb2a shows greater specificity than the ubiquitous ldb1a, ldb1b, or ldb2b (S2 Fig. and data retrieved from the Zebrafish Information Network (ZFIN) [30]). At 15 hours post fertilisation (hpf), ldb2a is present in the notochord and the lateral mesoderm, which gives rise to haematopoietic, endothelial, and pronephric derivatives (S2A Fig.). At 26 hpf, ldb2a expression continues in and around the blood vessels (S2B Fig.). Maternal/zygotic ldb2a is expressed ubiquitously throughout cleavage and blastula stage (0–4.7 hpf) embryos (S2C–S2F Fig.), but immediately before and during gastrulation (4.7–10 hpf), ldb2a becomes more specific in the yolk syncytial layer (YSL) (Figs. 2A and S2F, white arrowheads), an important source of Nodal signalling crucial for the specification of gastrula germ layers. This suggests a possible role for Ldb2a in signalling by this TGFβ superfamily member, we therefore initially focussed our studies on the function of Ldb2a in Nodal signalling during gastrula embryonic development.

Bottom Line: Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback.In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP.Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

View Article: PubMed Central - PubMed

Affiliation: Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.

ABSTRACT
As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

Show MeSH
Related in: MedlinePlus