Modulation of the Promoter Activation Rate Dictates the Transcriptional Response to Graded BMP Signaling Levels in the Drosophila Embryo

Abstract

Summary Morphogen gradients specify cell fates during development, with a classic example being the bone morphogenetic protein (BMP) gradient’s conserved role in embryonic dorsal-ventral axis patterning. Here, we elucidate how the BMP gradient is interpreted in the Drosophila embryo by combining live imaging with computational modeling to infer transcriptional burst parameters at single-cell resolution. By comparing burst kinetics in cells receiving different levels of BMP signaling, we show that BMP signaling controls burst frequency by regulating the promoter activation rate. We provide evidence that the promoter activation rate is influenced by both enhancer and promoter sequences, whereas Pol II loading rate is primarily modulated by the enhancer. Consistent with BMP-dependent regulation of burst frequency, the numbers of BMP target gene transcripts per cell are graded across their expression domains. We suggest that graded mRNA output is a general feature of morphogen gradient interpretation and discuss how this can impact on cell-fate decisions.

CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos

“…The two-step CRISPR approach uses two cut sites to first insert an attP ΦC31 phage recombination site into the gene region of interest. In the second step, the attP ΦC31 recombination site is used to integrate MS2 stem-loops and the DNA sequences that were initially removed in step 1 ( Figure 3 B) ( Baena-Lopez et al., 2013 ; Hoppe et al., 2020 ). Note: If the protocols described here are to be used to insert a protein tag, the exact position of the tag can be chosen by removing a region from the genome using two gRNAs, whereas a single cut site will place the tag sequences directly adjacent to the cut site and therefore be limited by gRNA availability.…”

“…Tagging an endogenous locus with MS2 sequences will enable live imaging studies to investigate and quantitate nascent transcription dynamics. For our recent study investigating the transcriptional regulation of endogenous Bone Morphogenetic Protein target genes see Hoppe et al., 2020 .…”

“…This can facilitate live imaging of nascent transcription in Drosophila . For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020) .…”

Live imaging and quantitation of nascent transcription using the MS2/MCP system in the Drosophila embryo

“…We also describe an analysis pipeline to extract and subsequently quantify transcription dynamics. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020) .…”

“…For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020) .…”