The embryo as a laboratory: quantifying transcription in Drosophila

Abstract

Transcriptional regulation of gene expression is fundamental to most cellular processes, including determination of cellular fates. Quantitative studies of transcription in cultured cells have led to significant advances in identifying mechanisms underlying transcriptional control. Recent progress allowed implementation of these same quantitative methods in multicellular organisms to ask how transcriptional regulation unfolds both in vivo and at the single molecule level in the context of embryonic development. Here we review some of these advances in early Drosophila development, which bring the embryo on par with its single celled counterparts. In particular, we discuss progress in methods to measure mRNA and protein distributions in fixed and living embryos, and we highlight some initial applications that lead to fundamental new insights about molecular transcription processes. We end with an outlook on how to further exploit the unique advantages that come with investigating transcriptional control in the multicellular context of development.

Keywords: embryogenesis; gene regulatory networks; live imaging; quantitative biology; single molecule FISH.

Figure 1. Cells in different contexts: culture vs. embryo

(A) Cells in culture require careful preparation to assure reproducibility across experiments. Female flies naturally prepare embryos in a high-throughput and reproducible manner. (B) Culturing allows examination of gene regulatory network behavior across a range of conditions in a synthetic environment. As such, it is not a priori evident which part of observed cell-to-cell variability (which can range between 50% and 500%) is due to sample preparation versus inherent fluctuations in the system. Studies of embryos reveal the degree of natural variability in gene expression that is tolerated by an intact system. As a system, the embryo is designed to generate a specific gene expression pattern within tight temporal and spatial constraints. This tight control results in levels of gene expression that can be reproducible within 10% and boundary positions with a 1% egg length reproducibility. (C) The absence of membranes between nuclei in the early embryo allows for the mixing of regulatory factors by diffusion. (D) Synchrony of the cell cycle is hardwired in the early fly developmental program. (E) Titrating input concentrations of transcription factors in cell culture requires the implementation of synthetic inducible circuits in multiple experiments. By contrast, transcription factor gradients in the early embryo span the natural range of patterning dynamics required for proper development.

Figure 2. Visualizing the central dogma in the embryo

(A) Developmental decisions can be assessed at multiple stages of the central dogma: from nascent mRNA transcript formation (and thus the number of active RNA polymerases loaded on the promoter), to cytoplasmic mRNA and protein distributions. (B) Methods to measure mRNA and protein distributions during development in fixed and living embryos (see text).

Figure 3. Signatures for the transcriptional basis of pattern formation in the fly embryo

(A) The variability in the instantaneous amount of mRNA being produced at sites of nascent transcript formation is as high as 50%. However, the mRNA molecules produced in different nuclei are averaged in the common cytoplasm of the syncytium, effectively reducing variability in the distribution of cytoplasmic mRNA molecules to 10%. (B) The variability in mRNA production observed in nuclei in (A) suggests a mechanism of transcription where the promoter switches between an ON and an OFF state. The molecular nature of this mechanism could be associated with enhancer-promoter looping or transient changes in chromatin accessibility, for example. (C) Live mRNA production monitoring reveals that patterns are formed by two serial steps of transcriptional regulation in single cells. First, a locus of a given gene makes a random binary decision whether to turn ON or not, with the local concentration of activator biasing this decision. Loci that are OFF will remain so for the whole interphase.By contrast, loci that turn ON will produce mRNA at a rate that in modulated by the local concentration of activator in an analog fashion. It is the combination of these two regulatory strategies that leads to macroscopic patterns of expression throughout the embryo.