br Experimental Procedures See also Supplemental Experimenta

Experimental Procedures See also Supplemental Experimental Procedures.
Author Contributions
Acknowledgments We thank Dr. Yun Deng for providing Tg (myl7:EGFP) zebrafish, Dr. Niels C. Bols and Dr. Peng Hu for their technical support with zebrafish. This work was supported by the National Natural Science Foundation of China (31571280, 31272648, and 31471182), the National Key Basic Research project (2010CB126306), National Key Technologies R&D Program (2012BAD26B01), Hubei Province Science and Technology project, and the Chinese 111 project.
Introduction Recent advances in exploring the molecular mechanisms of pluripotency revealed major differences between mice and other mammals (Manor et al., 2015; Nichols and Smith, 2009). Mouse embryonic stem cells (ESCs) self-renew in the naive state of pluripotency, a state characterized by permissiveness to single-cell dissociation, inhibiting differentiation by interleukin-6 family members, including leukemia inhibitory factor (LIF), stabilizing self-renewal after inhibiting MEK signaling, a transcriptome close to that of the epiblast of the pre- and peri-implantation blastocyst, and the capacity to participate in forming the three germ layers and generate germline chimeras on injection into the blastocelic cavity (Nichols and Smith, 2009). Conversely, ESCs generated from human and monkey pre-implantation embryos self-renew in the primed state of pluripotency as they express lineage markers and appear closer to commitment to differentiation (Nichols and Smith, 2009). The transcriptome of primate ESCs resembles that of EpiSC lines generated from the epiblast of the mouse post-implantation embryo (Brons et al., 2007; Tesar et al., 2007), a pluripotent cell layer that forms before the onset of gastrulation. They also have similar growth requirements. Both primate ESCs and mouse EpiSCs require fibroblast growth factor 2 (FGF2) and transforming growth factor (TGF-β) superfamily factors to inhibit differentiation, and MEK inhibition fails to stabilize self-renewal. Similar to EpiSCs in mice (Tesar et al., 2007), monkey ESCs also did not generate chimeras after an injection in a MAPK Inhibitor Library (Tachibana et al., 2012). Rabbit ESC lines were generated in several laboratories (Honda et al., 2008; Intawicha et al., 2009; Osteil et al., 2013; Tancos et al., 2012; Wang et al., 2006). These lines exhibited the cardinal features of pluripotency including long-term self-renewal, differentiation into ectodermal, mesodermal, and endodermal derivatives, and the capacity to form teratomas after injection into immunocompromised mice. When cytogenetic studies were performed, they featured a normal chromosomal complement (N = 44) (Wang et al., 2006; Osteil et al., 2013). Similar to primate ESCs, rabbit ESCs appear to be inherently primed. They rely on FGF2 and Activin/nodal/TGF-β but not on LIF signaling for the maintenance of pluripotency (Honda et al., 2009; Osteil et al., 2013; Wang et al., 2006, 2008), and express transcription factors associated with primed pluripotency in rodents (Osteil et al., 2013; Schmaltz-Panneau et al., 2014). However, we found that rabbit ESCs differ from primate ESCs in two aspects (Osteil et al., 2013). First, they have a different morphology with a lower nuclear-to-cytoplasmic ratio, a characteristic usually associated with a more advanced state in development. Second, they possess a DNA-damage checkpoint in the G1 phase of the cell cycle, which is absent in mouse, monkey, and human ESCs, and only acquired during differentiation (Aladjem et al., 1998; Filipczyk et al., 2007; Fluckiger et al., 2006; Momcilovic et al., 2009). Whether the presence of the G1 checkpoint in rabbit ESCs reflects a fundamental difference in pre-implantation embryo development between primates and rabbits or whether rabbit ESCs self-renew even closer to commitment to differentiation than primate ESCs is unknown at this stage. Another key aspect of the biology of rabbit pluripotent stem cells (PSCs) involves induced PSCs (iPSCs). We reported that rabbit iPSCs do not share all defining characteristics of primed pluripotency. Albeit dependent on FGF2 for self-renewal, rabbit iPSCs express naive pluripotency markers at higher levels, the naive-specific distal enhancer of Oct4 is more active, and they can uniquely be propagated using single-cell dissociation with trypsin, unlike rbESCs. Some cells in rabbit iPSC populations can colonize the rabbit pre-implantation embryo (Osteil et al., 2013). Such differences between ESCs and iPSCs have not been described in mice and primates.