ABSTRACT
Polycomb repressive complex 2 (PRC2) mediates histone H3K27me3 methylation and the stable transcriptional repression of a number of gene expression programs involved in the control of cellular identity during development and differentiation. Here, we report on the generation and on the characterization of a zebrafish line harboring a null allele of eed, a gene coding for an essential component of the PRC2. Homozygous eed-deficient mutants present a normal body plan development but display strong defects at the level of the digestive organs, such as reduced size of the pancreas, hepatic steatosis, and a loss of the intestinal structures, to die finally at around 10-12 days post fertilization. In addition, we found that PRC2 loss of function impairs neuronal differentiation in very specific and discrete areas of the brain and increases larval activity in locomotor assays. Our work highlights that zebrafish is a suited model to study human pathologies associated with PRC2 loss of function and H3K27me3 decrease.
Subject(s)
Digestive System/metabolism , Homeostasis , Neurons/cytology , Polycomb Repressive Complex 2/deficiency , Zebrafish/metabolism , Animals , Animals, Genetically Modified , Behavior, Animal , Cell Differentiation , Gene Expression Regulation, Developmental , Histones/metabolism , Larva/metabolism , Liver/metabolism , Lysine/metabolism , Methylation , Motor Activity , Mutation/genetics , Neurons/metabolism , Organ Specificity , Polycomb Repressive Complex 2/metabolism , Protein Processing, Post-Translational , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Activator-Like Effector Nucleases/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
To investigate the role of maternal Activin-like factors in the preservation of stemness and mesendoderm induction, their effects were promoted and inhibited using synthetic human Activin A or SB-505124 treatments, respectively, before the maternal to zygotic transition (MZT). To study the role of zygotic Activin-like factors, SB-505124 treatment was also used after the MZT. Promoting the signaling intensity of maternal Activin-like factors led to premature differentiation, loss of stemness, and no mesendoderm malformation, while its alleviation delayed the differentiation and caused various malformations. Inhibition of the zygotic Activin-like factors was associated with suppressing the ndr1, ndr2, oct4 (pou5f3), mycb and notail transcription as well as differentiation retardation at the oblong stage, and a broad spectrum of anomalies in a dose-dependent manner. Together, promoting the signal intensity of maternal Activin-like factors drove development along with mesendodermal differentiation, while suppression of the maternal or zygotic ones maintained the pluripotent state and delayed differentiation.
Subject(s)
Zebrafish Proteins , Zebrafish , Activins/genetics , Activins/metabolism , Activins/pharmacology , Animals , Gene Expression Regulation, Developmental , Intracellular Signaling Peptides and Proteins/genetics , Mesoderm/metabolism , Nodal Signaling Ligands/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Activin-like factors control many developmental processes, including pluripotency maintenance and differentiation. Although Activin-like factors' action in mesendoderm induction has been demonstrated in zebrafish, their involvement in preserving the stemness remains unknown. To investigate the role of maternal Activin-like factors, their effects were promoted or blocked using synthetic human Activin A or SB-431542 treatments respectively until the maternal to zygotic transition. To study the role of zygotic Activin-like factors, SB-431542 treatment was also applied after the maternal to zygotic transition. The effect of the pharmacological modulations of the Activin/Smad pathway was then studied on the mRNA expressions of the ndr1, ndr2, tbxta (no tail/ntl) as the differentiation index, mych, nanog, and oct4 (pou5f3) as the pluripotency markers of the zebrafish embryonic cells as well as sox17 as a definitive endoderm marker. Expression of the target genes was measured at the 16-cell, 256-cell, 1K-cell, oblong, dome, and shield stages using the real-time quantitative polymerase chain reaction (RT-qPCR). Activation of the maternal Activin signaling pathway led to an increase in zygotic expression of the tbxta, particularly marked at the oblong stage. In other words, promotion of the maternal Activin/Smad pathway induced differentiation by advancing the major peaks of ndr1 and nanog, thereby eliciting tbxta expression. Whereas suppression of the maternal or zygotic Activin/Smad pathway sustained the pluripotency by preventing the major peaks of ndr1 and nanog as well as tbxta encoding.
