Abstract
BackgroundConsistent 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.ResultsTo 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.ConclusionHDAC 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.
Highlights
Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis
The epigenetic machinery controlled by Histone Deacetylase (HDAC) is involved in LR patterning upstream of Nodal related 1 expression Xenopus embryos express a maternal form of HDAC mRNA that shows sequence homology to other HDACs [29]
Embryos in which the dominant negative (DN) HDAC mRNA was injected at the 1 cell stage exhibited a 17-fold increase in heterotaxia compared to controls (17% vs. 1% heterotaxia in control non injected embryos, n = 62, significant at p = 0.002), demonstrating that HDAC is functionally involved in embryonic LR patterning in Xenopus
Summary
Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. A different model focuses on much earlier stages, prior to gastrulation, when physiological events leverage asymmetry from the chirality of the intracellular cytoskeleton to set up asymmetrical movement of morphogens through cell fields [10,11,12]. One such morphogen is serotonin (5HT): a neurotransmitter of clinical relevance that has interesting roles outside the central nervous system [13]. It is known that 5HT accumulates in the right blastomeres in a rapid process dependent on asymmetric voltage gradients across the midline and the presence of open gap junctions through which it traverses [14,15,16]
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