Reprogramming and differentiation in mammals: motifs and mechanisms

Abstract

The natural reprogramming of the mammalian egg and sperm genomes is an efficient process that takes place in less than 24 hours and gives rise to a totipotent zygote. Transfer of somatic nuclei to mammalian oocytes also leads to their reprogramming and formation of totipotent embryos, albeit very inefficiently and requiring an activation step. Reprogramming of differentiated cells to induced pluripotent stem (iPS) cells takes place during a period of time substantially longer than reprogramming of the egg and sperm nuclei and is significantly less efficient. The stochastic expression of endogenous proteins during this process would imply that controlled expression of specific proteins is crucial for reprogramming to take place. The fact that OCT4, NANOG, and SOX2 form the core components of the pluripotency circuitry would imply that control at the transcriptional level is important for reprogramming to iPS cells. In contradistinction, the much more efficient reprogramming of the mammalian egg and sperm genomes implies that other levels of control are necessary, such as chromatin remodeling, translational regulation, and efficient degradation of no longer needed proteins and RNAs.

Figure 1. Time line for the development of Drosophila, Xenopus and Danio rerio in relation to the oocyte-to-embryo transition in the mouse

The time line is given in hours, with 0h fixed at fertilization. The time points when maternal message degradation (MMD), the mid-blastula transition (MBT) and embryonic genome activation (EGA) take place are indicated.

Figure 2. Mutation of the CPE in the Spin 4.1 kb 3'UTR leads to ineffective translation

Top panel – schematic representation of the Spin 4.1 kb reporter constructs used to determine the effect of mutation of the CPE on message stability in the FGO and ovulated oocyte. The CPE sequences in the Spin 4.1 kb transcript are indicated next to “intact 3'UTR”. The 3'CPE is 37 nt from the PAS, and the 5'CPE 119 nt away from the PAS. The sequences replacing the 3'CPE sequence in the Spin 4.1 kb transcript are indicated next to “mutation in CPE”. PAS indicates the polyadenylation signal sequence. Lower panel – Graphic representation of the decrease in translation of the reporter construct when the CPE sequence in the Spin 4.1 kb transcript is mutated. FGO – full-grown oocyte; FGO -> OO – transition from FGO to ovulated oocyte; OO – ovulated oocyte. Graph of reporter construct containing the intact CPE sequence; Graph of reporter construct containing the mutated CPE sequence.

Figure 3. Expression of “iPS” genes in oocytes and preimplantation embryos

RT-PCR, using cDNA from 2 oocyte or embryo equivalents, was used to determine the expression of iPS genes during the oocyte-to-embryo transition and in later preimplantation stages. The names of the genes are indicated on the right; oocyte and embryo stages are indicated at the top. FGO - full-grown oocyte; OO - ovulated oocyte; Zyg - zygote; e2C - early 2-cell stage; L2C - late 2-cell stage; 4/8C - 4-to-8-cell stage; Mor - morula; Blast – blastocyst. Primer information, PCR conditions, and results are available in the Gene Expression Database (GXD) (http://www.informatics.jax.org; J:138685).