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Histone deacetylase activity is necessary for left-right patterning during vertebrate development.

Carneiro K, Donnet C, Rejtar T, Karger BL, Barisone GA, Díaz E, Kortagere S, Lemire JM, Levin M - BMC Dev. Biol. (2011)

Bottom Line: To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery.The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator.The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology Center for Regenerative and Developmental Biology Tufts University, Medford, MA 02155 USA.

ABSTRACT

Background: Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known.

Results: To understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3's biological activity is dependent on 5HT binding.

Conclusion: HDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

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Mad3 is a new left-right determinant. (A) Single ventral left or ventral right blastomeres were injected with EngMad3 or Vp16Mad3 constructs and organ placement was scored at stage 45. Significant levels of heterotaxia were observed when embryos were injected with EngMad3 (left: 21%, n = 96, p < 0.001; right: 12%, n = 91, p = 0.005; control: Eng) or with Vp16Mad3 on the right side (17% n = 90, p < 0.001; 6% n = 127, p = 0.1, control: Vp16). (B) Embryos injected with EngMad3 or Vp16Mad3 were processed for in situ hybridization at stage 21 with a XNr-1 probe. XNr-1 misexpression was correlated with Vp16Mad3 injection (right injected embryos: 19% bilateral expression, n = 117; left injected: 5% bilateral expression, n = 105) and EngMad3 (left injected: 75% no expression, n = 74; right injected: 21% no expression, n = 93) if compared to control embryos (uninjected, 10% no expression, n = 103). (B') XNr-1 expression pattern characterized in EngMad3 or Vp16Mad3 injected embryos. I- Left expression indicated by the white arrow; II-right expression indicated by the gray arrow; III- Bilateral XNr-1 expression indicated by the two blue arrows; IV- Absence of XNr-1 expression as indicated by the two black arrows.
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Figure 5: Mad3 is a new left-right determinant. (A) Single ventral left or ventral right blastomeres were injected with EngMad3 or Vp16Mad3 constructs and organ placement was scored at stage 45. Significant levels of heterotaxia were observed when embryos were injected with EngMad3 (left: 21%, n = 96, p < 0.001; right: 12%, n = 91, p = 0.005; control: Eng) or with Vp16Mad3 on the right side (17% n = 90, p < 0.001; 6% n = 127, p = 0.1, control: Vp16). (B) Embryos injected with EngMad3 or Vp16Mad3 were processed for in situ hybridization at stage 21 with a XNr-1 probe. XNr-1 misexpression was correlated with Vp16Mad3 injection (right injected embryos: 19% bilateral expression, n = 117; left injected: 5% bilateral expression, n = 105) and EngMad3 (left injected: 75% no expression, n = 74; right injected: 21% no expression, n = 93) if compared to control embryos (uninjected, 10% no expression, n = 103). (B') XNr-1 expression pattern characterized in EngMad3 or Vp16Mad3 injected embryos. I- Left expression indicated by the white arrow; II-right expression indicated by the gray arrow; III- Bilateral XNr-1 expression indicated by the two blue arrows; IV- Absence of XNr-1 expression as indicated by the two black arrows.

Mentions: To probe the requirement of Mad3 between the left and right side of the embryo, the single dorsal or ventral, left or right blastomeres at the stage 3 (4 cells) were injected with mRNA encoding EngMad3 or Vp16Mad3 along with the lineage tracer lacZ mRNA. The embryos were then scored for organ placement at stage 45. Whereas dorsal injections led to considerable toxicity, ventral left or right expression of the EngMad3 (repressive) construct induced heterotaxia at a statistically significant level when compared to control Eng-expressing embryos (Figure 5A). Conversely, embryos injected with Vp16Mad3 in the right ventral blastomere presented significant heterotaxia when compared with Vp16 control injected embryos (Figure 5A).


Histone deacetylase activity is necessary for left-right patterning during vertebrate development.

Carneiro K, Donnet C, Rejtar T, Karger BL, Barisone GA, Díaz E, Kortagere S, Lemire JM, Levin M - BMC Dev. Biol. (2011)

Mad3 is a new left-right determinant. (A) Single ventral left or ventral right blastomeres were injected with EngMad3 or Vp16Mad3 constructs and organ placement was scored at stage 45. Significant levels of heterotaxia were observed when embryos were injected with EngMad3 (left: 21%, n = 96, p < 0.001; right: 12%, n = 91, p = 0.005; control: Eng) or with Vp16Mad3 on the right side (17% n = 90, p < 0.001; 6% n = 127, p = 0.1, control: Vp16). (B) Embryos injected with EngMad3 or Vp16Mad3 were processed for in situ hybridization at stage 21 with a XNr-1 probe. XNr-1 misexpression was correlated with Vp16Mad3 injection (right injected embryos: 19% bilateral expression, n = 117; left injected: 5% bilateral expression, n = 105) and EngMad3 (left injected: 75% no expression, n = 74; right injected: 21% no expression, n = 93) if compared to control embryos (uninjected, 10% no expression, n = 103). (B') XNr-1 expression pattern characterized in EngMad3 or Vp16Mad3 injected embryos. I- Left expression indicated by the white arrow; II-right expression indicated by the gray arrow; III- Bilateral XNr-1 expression indicated by the two blue arrows; IV- Absence of XNr-1 expression as indicated by the two black arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Mad3 is a new left-right determinant. (A) Single ventral left or ventral right blastomeres were injected with EngMad3 or Vp16Mad3 constructs and organ placement was scored at stage 45. Significant levels of heterotaxia were observed when embryos were injected with EngMad3 (left: 21%, n = 96, p < 0.001; right: 12%, n = 91, p = 0.005; control: Eng) or with Vp16Mad3 on the right side (17% n = 90, p < 0.001; 6% n = 127, p = 0.1, control: Vp16). (B) Embryos injected with EngMad3 or Vp16Mad3 were processed for in situ hybridization at stage 21 with a XNr-1 probe. XNr-1 misexpression was correlated with Vp16Mad3 injection (right injected embryos: 19% bilateral expression, n = 117; left injected: 5% bilateral expression, n = 105) and EngMad3 (left injected: 75% no expression, n = 74; right injected: 21% no expression, n = 93) if compared to control embryos (uninjected, 10% no expression, n = 103). (B') XNr-1 expression pattern characterized in EngMad3 or Vp16Mad3 injected embryos. I- Left expression indicated by the white arrow; II-right expression indicated by the gray arrow; III- Bilateral XNr-1 expression indicated by the two blue arrows; IV- Absence of XNr-1 expression as indicated by the two black arrows.
Mentions: To probe the requirement of Mad3 between the left and right side of the embryo, the single dorsal or ventral, left or right blastomeres at the stage 3 (4 cells) were injected with mRNA encoding EngMad3 or Vp16Mad3 along with the lineage tracer lacZ mRNA. The embryos were then scored for organ placement at stage 45. Whereas dorsal injections led to considerable toxicity, ventral left or right expression of the EngMad3 (repressive) construct induced heterotaxia at a statistically significant level when compared to control Eng-expressing embryos (Figure 5A). Conversely, embryos injected with Vp16Mad3 in the right ventral blastomere presented significant heterotaxia when compared with Vp16 control injected embryos (Figure 5A).

Bottom Line: To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery.The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator.The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology Center for Regenerative and Developmental Biology Tufts University, Medford, MA 02155 USA.

ABSTRACT

Background: Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member Nodal related 1 (Nr1) in the left lateral mesoderm plate is a highly conserved step regulating the situs of the heart and viscera. In Xenopus, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of Nr1. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known.

Results: To understand how an early physiological gradient is transduced into a late, stable pattern of Nr1 expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked Nr1 expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of Nr1 expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the Nr1 gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of Nr1 transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3's biological activity is dependent on 5HT binding.

Conclusion: HDAC activity is a new LR determinant controlling the epigenetic state of Nr1 from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.

Show MeSH
Related in: MedlinePlus