Abstract
SummarySelf-renewal circuitry in embryonic stem cells (ESCs) is increasingly defined. How the robust pluripotency program is dissolved to enable fate transition is less appreciated. Here we develop a forward genetic approach using haploid ESCs. We created libraries of transposon integrations and screened for persistent self-renewal in differentiation-permissive culture. This yielded multiple mutants in the Fgf/Erk and GSK3/Tcf3 modules known to drive differentiation and in epigenetic modifiers implicated in lineage commitment. We also identified and validated factors not previously considered. These include the conserved small zinc finger protein Zfp706 and the RNA binding protein Pum1. Pum1 targets several mRNAs for naive pluripotency transcription factors and accelerates their downregulation at the onset of differentiation. These findings indicate that the dismantling of pluripotent circuitry proceeds at multiple levels. More broadly they exemplify the power of haploid ESCs for genetic interrogation of developmental processes.
Highlights
Rodent embryonic stem cells (ESCs) exhibit the identity and pluripotency of naive preimplantation epiblast cells with the additional attribute of extended self-renewal (Nichols and Smith, 2012)
A Haploid ESC Screen to Identify Genes that Promote Exit from Ground State Self-Renewal To isolate and analyze mutant ESCs impeded in progression from self-renewal, we used a haploid reporter cell line (HRex1GFPd2) in which a destabilized version of GFP is expressed from the endogenous Rex1 (Zfp42) locus (Wray et al, 2011)
Mutagenesis of a haploid genome will result in homozygous diploid mutant ESCs
Summary
Rodent ESCs exhibit the identity and pluripotency of naive preimplantation epiblast cells with the additional attribute of extended self-renewal (Nichols and Smith, 2012). The molecular machinery and underlying genetic circuitry that sustain ESC character during self-renewal have been extensively characterized (Young, 2011). Less studied is the process by which ESCs exit the naive state to embark upon differentiation. In contrast to the ordered program of germ layer segregation that unfolds deterministically in the embryo and is obeyed by ESCs in chimeras, differentiation in vitro is asynchronous and disorganized (Lowell et al, 2006). A timely opportunity for application of forward genetics to dissect this complex developmental transition arises from the recent derivation of haploid mouse ESCs (Elling et al, 2011; Leeb and Wutz, 2011)
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