Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation

Abstract

In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, “classical” multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms.

Introns Control Stochasticity in Metazoan Gene Expression

“…To quantify intrinsic noise in animals expressing alleles with and without introns, we conducted quantitative microscopy as we have previously described (28). We measured mEGFP and mCherry signals from individual cells in our field of view using a confocal microscope, Fig.…”

“…We then measured the expression levels of the intron-bearing and intronless sets of reporter alleles, which were expressed from the exact same locus (tt5605 on Chromosome II), using a confocal microscope ( Fig. 3B), as we described previously 32 . We quantified allele bias in individual worm intestine cells as intrinsic noise, visible as deviation from the 45° central trendline on scatter plots, using the same analytical framework as prior reports quantifying intrinsic noise in bacteria 9 and yeast 8 cells.…”

Developmental robustness in the Caenorhabditis elegans embryo

“…The quantity of elt‐2 mRNA appears to be relatively constant per cell in the early embryo (Raj et al, ). Although variation in elt‐2 expression has not been examined in adults, expression of a single‐copy reporter for hsp‐16.2 in the intestine is surprisingly similar from animal to animal (75–88% correlation) (Mendenhall et al, ), suggesting that variation in gene expression is generally low in the adult gut. Hence, C. elegans may not have evolved mechanisms to accommodate variation in gut gene expression, relying instead on upstream components to assure consistency in elt‐2 expression.…”

“…Although the proposed structure of the C. elegans endoderm GRN has remained largely unchanged in over a decade, there is still much to be learned. Newer methods allow: Quantitative measurements to be made of gene expression at the level of individual mRNAs (Raj et al, ) and protein (Raj et al, ; Tanenbaum et al, ; Mendenhall et al, ), semi‐automatic embryonic‐cell lineage tracing using fluorescent reporters (Bao et al, ), definition of the transcriptome of individual embryonic cells (Hashimshony et al, ), and high‐resolution analysis of the roles of specific cis ‐regulatory sites following the introduction of promoter mutations into genes in their native chromosomal context (Waaijers and Boxem, ). These newer approaches could be used in combinations to probe gene expression variation under normal conditions and as a result of specific network perturbations; thus, it should be possible to determine the roles of all the known factors that control variation at the level of gene products and in overall phenotype.…”

hsp-16.2 chaperone biomarkers track physiological states of proteome dosage

“…We focused on gene expression in the intestine cells of adult animals because that is the tissue that makes the most signal for hsp-16.2 reporters 13,16,17 , and because it is the point in life we used to predict lifespan and thermotolerance 12,13 , and because we had developed technical methods for in vivo reproducible quantification of gene expression in single intestine cells 16 .…”