Intracellular expression profiles measured by real-time PCR tomography in the Xenopus laevis oocyte

Abstract

Real-time PCR tomography is a novel, quantitative method for measuring localized RNA expression profiles within single cells. We demonstrate its usefulness by dissecting an oocyte from Xenopus laevis into slices along its animal–vegetal axis, extracting its RNA and measuring the levels of 18 selected mRNAs by real-time RT-PCR. This identified two classes of mRNA, one preferentially located towards the animal, the other towards the vegetal pole. mRNAs within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. The polarization is substantial, though not extreme, with around 5% of vegetal gene mRNA molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. Most animal pole mRNAs were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mRNA expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mRNA gradients.

Single blastomere expression profiling of Xenopus laevis embryos of 8 to 32-cells reveals developmental asymmetry

“…As markers to probe the formation of the three developmental axes we chose ten maternal transcripts that have been implicated in the dorso-ventral patterning: dvl2, dvl3, lrp6, wnt11, tcf3, gsk3b, ctnnb1( = β -catenin), foxh1, trim36 and axin1 16192021, one important marker for left-right specification: vg1 18, and nine genes implicated in animal-vegetal orientation: dazl, cdx1 ( = xcad2), wnt11, vg1, vegt, trim36, ddx25 ( = deadsouth), otx1, and maml1 9192223. We also included 25 mRNAs that have previously been observed in the mature oocyte: fzd7, bmp2, pias1, foxr1, frat1, mapk8, odc1, 18S rRNA, 5S rRNA, cyc1, acta, tubb, gapdh, eef1a1, RNA polymerase II, U3 snoRNA, par1, oct60, est1, apc, zpc, mos, stat3, and pax6 89.…”

“…The germ layers, endoderm, mesoderm, and ectoderm, are formed along the animal-vegetal axis such that the vegetal part of the oocyte becomes the endoderm and the most distal animal part of the oocyte gives rise to the ectoderm2526. Transcripts of genes coding specific endodermal and mesodermal factors, such as vg1 , wnt11, and vegt, are localized in the vegetal hemisphere8922. Genes responsible for primordial germ cell formation, such as dazl and cdx1, are localized towards the extreme vegetal pole9.…”

“…These were transcribed during oogenesis from solely maternal chromosomes. The maternal mRNAs are asymmetrically distributed along the animal-vegetal axis of the oocyte and direct the specialization of the animal and vegetal parts789. The second embryonic body axis is formed after fertilization and separates the embryo into a dorsal and a ventral part.…”

“…We have previously used quantitative real-time PCR (qPCR) expression profiling of cryostat sections (qPCR tomography) of a single oocyte to identify transcripts that form gradients along the animal-vegetal axis89. These transcripts will become differentially distributed among the animal and the vegetal blastomeres formed at the third cell division.…”

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

“…It has the potential to have a major impact on molecular analyses ranging from clinical applications, such as biomarker analysis (2 ), viral detection (3 ), prognostic monitoring (4 ), and fetal screening (5 ), to research applications such as phage-host interactions (6 ) and intracellular profiling (7 ). dPCR can also be applied to assist with the library preparation needed for massively parallel (or next generation) sequencing methods (8 ).…”