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

In agreement with our result indicating that the HDAC block leads to an increase in histone H4 and H3 acetylation levels, Mad signaling was shown to be important in the control of global levels of chromatin structure also characterized by acetylation of heterochromatin regions. Indeed, Mad proteins have been shown to coordinate chromatin modifications resulting in a significant decrease in acetylated histones H3 and H4 in cell culture [71].

A key feature of 5HT signaling during Xenopus LR development is that it functions during cleavage stages, prior to mid-blastula transition (MBT), and thus largely under zygotic transcriptional silence. It was previously shown that early 5HT signaling is important to the correct placement of Xnr-1 expression on the left side of embryo [15]. Indeed, constitutive repressive form of Mad3 protein (EngMad3) can induce heterotaxia and blocks Xnr-1 expression on the left side (Figure 5), which indicates its endogenous role as a repressor of Xnr-1. On the other hand, Vp16Mad3 injections could relieve the repressive state of Xnr-1 on the right side leading to its ectopic expression at stage 21 (Figure 5). In contrast to transcriptional control of Xnr-1, we propose that the EngMad3 phenotype is due to a constitutive recruitment of repressive elements of the cell machinery that can lead to a repressive state of the chromatin that can be maintained throughout development. Conversely, the Vp16Mad3 phenotype is compatible with an open chromatin structure leading to the ectopic induction of Xnr-1. Interestingly, injections with the HDAC DN and Vp16Mad3 on the right and HDAC WT and EngMad3 on the left gave rise to consistent heterotaxia, indicating that Mad3 and HDAC functions during LR establishment take place in the same subset of blastomeres and may converge on Nr1. This hypothesis is corroborated by the presence of 2 putative Mad binding sites CANNTG [72] in the intronic region of Nr1 (Figure 3A2). Interestingly, the second site is placed in the region that contains the FAST binding sites that are important to proper control the asymmetric expression of Nr1 in the left side of the embryo [53]. This interesting feature suggests that Mad protein could bind to this region of the Nr1 gene.

This analysis, along with the established status of Mad3 as a very well-characterized partner for HDACs, indicates that 5HT/Mad3 signaling could couple to repressive elements belonging to the epigenetic machinery of the early embryo. In addition, HDAC and Mad3 mRNA symmetric expression patterns argue in favor of a symmetric distribution for both proteins. In this context, we hypothesize that 5HT binding on Mad3 would be an asymmetric signal important to confer specificity for HDAC activity in the context of LR development, decreasing the levels of histone acetylation on the Nr1's intronic region (Figure 7).

Indeed, a recent study has demonstrated an antidepressant-like effect for HDACs inhibitors similar to the effects of fluoxetine, an effective inhibitor for 5HT re-uptake, [73] that randomizes LR asymmetry [16]. These data suggest a synergistic role for 5HT and HDAC pathways, indicating they may be acting in the same pathway and reinforcing our model that brings together 5HT and epigenetic machinery.

In Xenopus and many invertebrates, consistent asymmetry is determined by very early biophysical and physiological events taking place long before asymmetric gene expression and ciliary flow [10,11]. While these early mechanisms are mapped onto different embryonic architectures in a variety of ways throughout phyla [74,75], left-sided Xnr-1 expression is a well-conserved regulator of the situs of the heart and visceral organs [76]. Our data on epigenetic modulation provide the first detailed glimpse into the molecular events that allow physiological events during very early stages to be solidified into cascades of gene expression.

Analysis of the mouse and Xenopus Nr-1 gene has revealed a regulatory sequence in the coding region that is crucial for the asymmetric expression at the LPM and that is targeted by the complex FAST/SMADS [53,77]. This asymmetric enhancer (ASE) sequence is present in the intronic region [53]. The transcription factor FAST-1 mediates TGFβ signaling, and, together with SMAD-2 and -4, has been shown to be necessary to trigger Xnr-1 asymmetric expression by binding to the Xnr-1 ASE in the LPM [77]. However, all signaling molecules that play a role in the asymmetric expression of Nr-1 characterized so far are symmetrically expressed in the LPM, including the immediate upstream player FAST-1 [77]. Thus, it becomes crucial to understand how upstream symmetric events taking place at the cellular levels result in reliably asymmetric Nr-1 expression. In addition, it is known that the right side of the embryo has an intrinsic ability to express Xnr-1, indicating that the cells on the right side have all machinery needed to express Xnr-1 but are normally repressed from doing so [78].

Our results show that the Xnr-1 intronic region contains high levels of acetylated histone H3 and H4 and H3K4me2 after NaB treatment (Figure 3) and this correlates with absence of Xnr-1 expression. Although the biological significance in terms of transcriptional outcomes due to H3K4me2 is still under debate, it is becoming clear that this epigenetic marker may prevent aberrant gene expression or modulate transcriptional outcomes [55]. In the context of our results, a possible interpretation is that H3K4me2 could work as a repressive marker facilitating the efficiency of inhibition by Lefty. In normal embryos these results suggest that HDAC could target the Xnr-1 intronic region (ASE) early during development leading to a decrease in the levels of acetylated histones H3 and as a consequence preventing H2K4me2 from being deposited in this region, making this region accessible to FAST related proteins. By the time of Xnr-1 expression initiation, the absence of H3K4me2 would increase the efficiency of activation of Xnr-1 expression, which will not result in significant noise, leading to stable expression of Xnr-1.

Future experiments addressing the balance in acetylation and methylation levels of histones between left and right sides of the embryo will be necessary to understand how the epigenetic machinery controls different elements during LR determination besides Nr1. For instance, the investigation of epigenetic modifications on other left-right genes, such as Lefty and Pitx will be important to understand how global HDAC activity blockade changes the chromatin status and how these changes are transduced into different states of LR genes' activity. For example, Lefty is an inhibitor of Nr1 and any epigenetic change on Lefty due to HDAC blockade may also affect Nr1 expression, providing a possible explanation on the absence of XNr-1 expression when HDAC activity is blocked. This hypothesis is supported by the feed-forward and feedback loop between Nr1 and Lefty that is important to exclude Nodal from being expressed on the right side of the embryo. Consistent with this rationale, our data implicate HDAC activity as important to set Nr1 expression but also suggest that HDAC activity may target Lefty, leading to its ectopic expression on the left side that ultimately will repress Nr1 expression. For this reason, a comprehensive understanding of the epigenetic regulation of the key asymmetric genes, and the upstream components linking the sidedness of transcription to early physiological gradients, will be a crucial aspect of fleshing out a most fascinating aspect of left-right patterning.

HDAC activity is a new LR determinant controlling the transcription of the Xnr-1 gene. Molecular-genetic and pharmacological blockade of HDAC activity led to deposition of epigenetic markers on the Xnr-1 gene that was correlated with misexpression of Xnr-1 and organ heterotaxia. The known HDAC partner Mad3 is also a new functional LR determinant whose biological activity during LR establishment is dependent on 5HT. Taken together these data suggest a model in which epigenetic machinery transduces early physiological gradients into much later transcriptional effectors during establishment of consistent organ situs in vertebrate embryogenesis.

KC designed and carried out the experiments, and wrote the manuscript. BK, CD, and TR conceived and CD and TR carried out the Chromatographic 5HT affinity capture screening. GB and ED designed and GB carried out the Mad3 and 5HT in vitro interaction assay (Surface Plasmon Resonance assay). KC and ML interpreted the data and designed experiments. SK carried out the Mad3 modeling and simulation. JML designed and constructed many of the plasmids. ML conceived, designed, and wrote the manuscript. All authors read and approved the final manuscript.