Abstract
The appearance, progression, and potential role for circadian rhythms during early development have previously focused mainly on the suprachiasmatic nucleus (SCN) and peri- and postnatal expression of canonical clock genes. More recently, gene expression studies in embryonic stem cells have shown that some clock genes are expressed in undifferentiated cells; however rhythmicity was only established when cells are directed toward a neural fate. These studies also concluded that a functional clock is not present in ESCs, based solely on their gene expression. The null hypothesis underlying the present study is that embryonic stem cells become rhythmic in both clock gene expression and glucose utilization only when allowed to spontaneously differentiate. Undifferentiated stem cells (ESCs, n = 6 cultures/timepoint for all experiments) were either maintained in their pluripotent state or released into differentiation (dESCs, n = 6 cultures/timepoint for all experiments). Glucose utilization was assayed through 2-deoxyglucose uptake measurement, and clock gene and glucose transporter expression was assayed every 4 hours for 2 days in ESCs and dESCs by quantitative PCR (qPCR) in the same cell lysates. Undifferentiated stem cells expressed a self-sustained rhythm in glucose uptake that was not coincident with rhythmic expression of clock genes. This physiological rhythm was paralleled by glucose transporter mRNA expression. Upon differentiation, circadian patterns of some but not all clock genes were expressed, and the amplitude of the glucose utilization rhythm was enhanced in dESCs. These data provide the earliest evidence of a functional circadian clock, in addition to further challenging the idea that rhythmic transcription of clock genes are necessary for rhythmic physiological output and suggest a role for a clock-controlled physiology in the earliest stages of development.
Highlights
Circadian rhythms and the cellular/physiological processes they control have been established as a highly conserved mechanism by which organisms are able to anticipate daily changes in the environment and to temporally coordinate complex processes [1]
Stem Cell Morphology/identity ESCs maintained under Leukemia Inhibitory Factor (LIF) displayed morphology consistent with that of undifferentiated embryonic stem cells when imaged just before sampling (Fig. 1A)
Rhythmic glucose utilization precedes the development of the molecular clockworks in embryonic stem cells; clock gene
Summary
Circadian rhythms and the cellular/physiological processes they control have been established as a highly conserved mechanism by which organisms are able to anticipate daily changes in the environment and to temporally coordinate complex processes [1]. The nuclear receptors REVERB-A and ROR-a competitively bind retinoic acid-related orphan receptor response elements (RORE) on the mBmal promoter region and act to inhibit and activate mBmal transcription, respectively [3]. These nuclear receptors have been suggested as a crucial link between the circadian timekeeping mechanism and physiology [4]. This transcriptional/translational feedback loop is frequently referred to as the ‘‘canonical molecular circadian clock’’ [2]
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