The use of double-stranded (ds) RNA is a powerful way of interfering with gene expression in a range of organisms, but doubts have been raised about whether it could be successful in mammals. Here, we show that dsRNA is effective as a specific inhibitor of the function of three genes in the mouse, namely maternally expressed c-mos in the oocyte and zygotically expressed E-cadherin or a GFP transgene in the preimplantation embryo. The phenotypes observed are the same as those reported for null mutants of the endogenous genes. These findings offer the opportunity to study development and gene regulation in normal and diseased cells.
Leukemia inhibitory factor (LIF) activates the transcription factor signal transducer and activator of transcription 3 (STAT3), which results in the maintenance of mouse embryonic stem cells in the pluripotent state by inhibiting both mesodermal and endodermal differentiation. How the LIF/STAT3 pathway inhibits commitment to both mesoderm and endoderm lineages is presently unknown. Using a hormone-dependent STAT3 and with microarray analysis, we identified 58 targets of STAT3 including 20 unknown genes. Functional analysis showed that 22 among the 23 STAT3 target genes analyzed contribute to the maintenance of the undifferentiated state, as evidenced by an increase in the frequency of differentiated colonies in a self-renewal assay and a concomitant elevation of early differentiation markers upon knockdown. Fourteen of them, including Dact1, Klf4, Klf5, Rgs16, Smad7, Ccrn4l, Cnnm1, Ocln, Ier3, Pim1, Cyr61, and Sgk, were also regulated by Nanog. Analysis of lineage-specific markers showed that the STAT3 target genes fell into three distinct categories, depending on their capacity to inhibit either mesoderm or endoderm differentiation or both. The identification of genes that harness self-renewal and are downstream targets of both STAT3 and Nanog shed light on the mechanisms underlying functional redundancy between STAT3 and Nanog in mouse embryonic stem cells.
Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.
Gastrulation in rodents is associated with an increase in the rate of growth and with the start of differentiation within the embryo proper. In an effort to understand the role played by the cell cycle control in these processes, expression of cyclin D1, D2, and D3-three major positive regulators of the G1/S transitionhas been investigated by in situ hybrization and RT-PCR. Cyclin D1 and D2 transcripts are first detected in the epiblast at gastrulation, when a proliferative burst occurs, and subsequently in its differentiated derivatives within the embryo proper, indicating that activation of their expression takes place prior to the differentiation of epiblast progenitors. In contrast, cyclin D3 transcript is undetectable in the epiblast itself and its expression is activated exclusively in extraembryonic tissues of both epiblast and trophoblast origin. During neurulation, expression of each cyclin D RNA is dynamically regulated along the anterior-posterior axis. In the hindbrain, cyclin D1 and D2 show distinct segment-specific restricted expression and this pattern is conserved between mouse and chick. These results strongly suggest that D-type cyclins act as developmental regulators.
The proliferation rate and differentiation state were investigated in porcine inner cell masses (ICMs) and epiblasts in vitro. ICMs isolated from early blastocysts (Day 7 of pregnancy) and epiblasts isolated from preelongated blastocysts (Day 11 of pregnancy) were cultured for up to 5 days in the presence of human leukemia inhibitory factor (hLIF) (1000 U/ml). The proliferation rate was evaluated by determination of the percentage of cells in S-phase. The differentiation state was determined by studying the expression of the stage-specific embryonic antigen-1 (SSEA-1), a marker for undifferentiated murine embryonic stem (ES) cells, and the expression of laminin and cytokeratins 8/18, markers of ES cell differentiation. The staining pattern showed that freshly collected Day 11 epiblasts appeared undifferentiated but rapidly lost this characteristic in vitro. A decrease in the proliferation rate was also observed during culture. This decrease was reduced in the presence of high concentrations of hLIF (optimal concentrations: 5000 U/ml). Conversely, treatment of Day 11 epiblast cells with retinoic acid, an agent known to induce differentiation in murine ES cells, caused a dramatic decrease in the proliferation rate in vitro. In contrast to Day 11 epiblasts, Day 7 ICMs expressed SSEA-1 in vitro and showed a higher proliferation rate (p < 0.01). However, their proliferation rate also decreased during culture and following trypsinization. These results indicate that the undifferentiated characteristics of Day 7 ICMs are more likely to be maintained in vitro than are those of Day 11 epiblasts, which are rapidly committed into early differentiation.
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