Our model predicts such controls, because the HDAC mRNA is expressed symmetrically. thus, an asymmetric selleck bio signal is needed to confer the observed con sistent difference in HDAC activity on the L and R sides. Because rightward redistribution of maternal sero tonin during cleavage stages has been shown to be necessary for the establishment of LR asymmetry, we sought mechanisms by which 5HT could couple to the epigenetic machinery. We performed a high throughput proteomic assay to identify novel 5HT binding proteins present in the cytoplasmic fraction that may interact with known epige netic machinery. A resin conjugated with 5HT was used for affinity capture analysis from whole protein lysate of Xenopus laevis embryos at the 1 cell stage.

Two aliquots of 5HT conjugated resin were loaded with either frog embryo lysate plus 20 mM 5HT or frog embryo lysate. The eluate from both experi ments was subjected to a 10% SDS PAGE and, after proper sample preparation and trypsin in solution diges tion, the peptide mixtures were analyzed by LC MS MS. The raw data collected from the ion trap mass spec trometer was analyzed for protein identification using a database for Xenopus laevis. The screen identified 11 proteins as possible 5HT interactors. Because we were interested in mechanisms by which intracellular 5HT binding proteins could couple to the cytoplasmic epige netic machinery, we focused on candidate proteins with known interactions with HDAC proteins and or DNA binding domains. We did not recover the classical 5HT transmembrane receptors in this screen, since we focused our analysis on the cytoplasmic protein fraction.

Indeed, our screen success fully identified 10 candidates with characteristics of cytoplasmic proteins and 1 candidate having a DNA binding domain. This DNA binding protein was identi fied as the Max interacting transcriptional repressor Mad3. Mad3 is involved in the control of the epigenetic state of cells, since it can antagonize transcription by recruiting co repressor complexes that contain HDAC I. Indeed, the repressive activity of Mad proteins has been shown to be due to HDAC activity and inhibited by HDAC blockers. Mad proteins also interact with Max, and this complex has been implicated in different aspects of cell biology and cancer transformation.

Mad3 protein and mRNA are present in the early embryo A role for Mad3 as a new player in the 5HT signaling pathway during LR patterning requires that Mad3 protein be already present in early embryos. Indeed, Mad3 mRNA is present in the blastomeres at the 4 cell stage by in situ Batimastat hybridization. By the 32 64 cell stage, Mad3 mRNA is found only in the animal cells. To validate the presence of Mad3 protein in early embryos, as detected on the ion trap mass spectrometer, we performed two different sets of analysis.