The Regulatory Logic of a Dose-Dependent Fate Decision: How a Low-Amplitude Erk Input Patterns Abd-B Expression to Produce Drosophila Tail Structures

Abstract

Early in development, cells receive information about their location in an embryo through external cues, which they must interpret to output specific transcriptional responses required for the proper development of the body structures at each position. The same set of signaling cues can give rise to a range of cell fates depending on the specific timing and concentration of the signal, thus the delivery dynamics of an input are essential to inform a cell’s response. Despite their importance in development, these dynamics are difficult to study as small changes can have multi-faceted effects, and signaling pathways often involve complex feedback mechanisms and redundancy in regulation. However, the use of optogenetics to manipulate the amount of signal delivered through precisely timed light inputs offers a novel approach to investigate complicated regulatory networks and directly examine how signaling dynamics impact transcriptional outputs. One system in which these signaling dynamics are incredibly important, yet incompletely understood, is the terminal extracellular signal-regulated kinase (Erk) network in the Drosophila embryo. Early in development, a very low level of Erk activation patterns a narrow stripe of Abdominal-B (Abd-B) expression near the embryo’s posterior, which is required to form posterior spiracle “tail structures” in the adult fly. Abd-B expression requires tailless (tll), a direct Erk target gene and known transcriptional repressor, but it is unclear how this repressor mediates activation of Abd-B expression. In this thesis, I took advantage of the power of optogenetics to dissect the regulatory network that produces a stripe at the correct time in development and position along the embryo’s anterior-posterior axis. I applied specific optogenetic Erk inputs to embryos and then measured the effects on tll and Abd-B expression. I also built new MS2 biosensors for measuring endogenous Abd-B transcription in live embryos to characterize its expression dynamics in relation to tll. I found that Abd-B transcription is promoted by low levels of tll but repressed by higher levels of tll. To make sense of this tll concentration-dependent activation to repression switch, I used optogenetic stimulation to further examine the regulatory relationships between tll, Abd-B, and the gap genes, hypothesizing that a gap gene might be the missing link between a low level of tll expression and activation of Abd-B. I characterized the gap gene giant (gt) as a direct Abd-B repressor and found that expression of tll at even low levels silences gt expression. These findings informed a model for Abd-B regulation in which low levels of tll repress gt to promote Abd-B expression in a narrow stripe. The borders of the stripe are set through repression by gt at the anterior and high levels of tll at the posterior. Overall, this thesis offers a mechanism through which a precisely low level of a signaling input can produce a particular developmental fate.