Single-cell Gene Expression Analysis Research Articles

Single-cell quantitative RT-PCR analysis of Cpt1b and Cpt2 gene expression in mouse antral oocytes and in preimplantation embryos

Fatty acids represent an important energy source for preimplantation embryos. Fatty acids oxidation is correlated with the embryo oxygen consumption which remains relatively constant up to the 8-cell stage, but suddenly increases between the 8-cell and morula stages. The degradation of fatty acids occurs in mitochondria and is catalyzed by several carnitine acyl transferases, including two carnitine palmitoyl transferases, CPT-I and CPT-II. We have carried out a study to determine the relative number of transcripts of Cpt1b and Cpt2 genes encoding for m-CPT-I and CPT-II enzymes, during mouse preimplantation development. Here we show that Cpt1b transcripts are first and temporally detected at the 2-cell stage and reappear at the morula and blastocyst stage. Cpt2 transcripts decrease following fertilization to undetectable levels and are present again later at the morula stage. These results show that transcription of both Cpt1b and Cpt2 is triggered at the morula stage, concomitantly with known increasing profiles of oxygen uptake and fatty acids oxidation. Based on the number of Cpt2 transcripts detected, we could discriminate the presence of two groups of embryos with high and low number of transcripts, from the zygote throughout preimplantation development. To further investigate if the establishment of these two groups of embryos occurs prior to fertilization, we have analyzed the relative number of transcripts of both genes in antral and ovulated MII oocytes. As for preimplantation embryos, MII oocytes show two groups of Cpt2 expression. Antral oocytes, classified according to their chromatin configuration in SN (surrounded nucleolus, in which the nucleolus is surrounded by a rim of Hoechst-positive chromatin) and NSN (not surrounded nucleolus, in which this rim is absent), show three groups with different numbers of Cpt2 transcripts. All NSN oocytes have a number of Cpt2 transcripts doubled compared to that of the group of MII oocytes with high expression. Instead, SN oocytes could be singled out into two groups with high and low numbers of Cpt2 transcripts, similar to those found in MII oocytes. The results of this study point out a correlation between the timing of fatty acids oxidation during preimplantation development and the expression of two genes encoding two enzymes involved in the oxidative pathway. Furthermore, although the biological meaning for the presence of two groups of oocytes/embryos with different levels of Cpt2 transcripts remains unclear, the data obtained suggest a possible correlation between the levels of Cpt2 expression and embryo developmental competence.

Single-Cell Analysis of Regulatory Gene Expression in Quiescent and Activated Mouse Skeletal Muscle Satellite Cells

Repair and regeneration of adult skeletal muscle are mediated by satellite cells. In healthy muscle these rare mononucleate muscle precursor cells are mitotically quiescent. Upon muscle injury or degeneration, members of this self-renewing pool are activated to proliferate and then differentiate. Here we analyzed in single satellite cells the expression of a set of regulatory genes that are candidates for causal roles in satellite cell activation, maturation, and differentiation. Individual cells were identified as satellite cells and selected for analysis based on their physical association with single explanted myofibers or their position beneath the basal lamina in unperturbed muscle tissue. Using a multiplex single-cell RT-PCR assay we simultaneously monitored expression of all four MyoD family regulators of muscle determination and differentiation (MRFs) together with two candidate markers of satellite cell identity, c-met and m-cadherin. By making these measurements on large numbers of individual cells during the time course of satellite cell activation, we were able to define which expression states (possible combinations of the six genes) were represented and to specify how the representation of each state changed with time. Activated satellite cells began to express either MyoD or myf5 first among the MRFs; most cells then expressed both myf-5 and MyoD simultaneously; myogenin came on later in cells expressing both MyoD and myf5; and many cells ultimately expressed all four MRFs simultaneously. The results for fiber-associated satellite cells from either predominantly fast or slow muscles were indistinguishable from each other. The c-met receptor tyrosine kinase was also monitored because it is a candidate for mediating activation of quiescent satellite cells (Allenet al.,1995) and because it might also be a candidate molecular marker for satellite cells. A significant difficulty in studying mouse satellite cells has been the absence of molecular markers that could identify them in the quiescent state before expression of MRFs or desmin and distinguish them from fibroblasts. We show here that c-met receptor is present beneath the basal lamina on presumptive satellite cells in intact muscle and that c-met mRNA and protein are expressed by all myofiber-associated satellite cells from the time of explant through the course of activation, proliferation, and differentiation. c-met was not detected in muscle-derived fibroblasts or in other mononucleate cells from healthy muscle explants. When compared directly with m-cadherin, which has previously been suggested as a marker for quiescent satellite cells, m-cadherin mRNA was detected only in a small subset of satellite cells at early times after myofiber explant. However, at late times following activation (by 96 hr in this fiber culture system), c-met and m-cadherin were uniformly coexpressed. From the individual satellite cell expression types observed, a model of the satellite cell population at rest and during the time course of activation was generated.

Single-cell analysis of gene expression in the nervous system. Measurements at the edge of chaos.

The characteristic functions of tissues and organs result from the integrated activity of individual cells. Nowhere is this more evident than in the nervous system, where the activities of single neurons communicating via electrical and chemical signals mediate complex functions, such as learning and memory. The past decade has seen an explosion in the identification of genes encoding proteins, such as voltage-gated channels and neurotransmitter receptors, responsible for neuronal excitability. These studies have highlighted the fact that even within a neuroanatomically defined region, the coexistence of multiple cell types makes it difficult, if not impossible, to correlate patterns of gene expression with function. The recent development of techniques sensitive enough to study gene expression at the single-cell level promises to break this bottleneck to our further understanding. Using examples taken from our own laboratories and the work of others, we review these techniques, their application, and discuss some of the difficulties associated with the interpretation of the data.