Multimodal transcriptional control of pattern formation in embryonic development

Abstract

Predicting developmental outcomes from regulatory DNA sequence and transcription 17 factor patterns remains an open challenge in physical biology. Using stripe 2 of the even-skipped 18 gene in Drosophila embryos as a case study, we dissect the regulatory forces underpinning a key 19 step along the developmental decision-making cascade: the generation of cytoplasmic mRNA 20 patterns via the control of transcription in individual cells. Using live imaging and computational 21 approaches, we found that the transcriptional burst frequency is modulated across the stripe to 22 control the mRNA production rate. However, we discovered that bursting alone cannot 23 quantitatively recapitulate the formation of the stripe, and that control of the window of time over 24 which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical 25 modeling revealed that these regulatory strategies-bursting and the time window-obey different 26 kinds of regulatory logic, suggesting that the stripe is shaped by the interplay of two distinct 27 underlying molecular processes. 28 29 35the regulatory logic of the enhancer elements that dictate the behavior of these networks, the 36 precise prediction of how gene expression patterns and developmental outcomes are driven by 37 transcription factor concentrations remains a central challenge in the field (Vincent et al., 2016). 38 Predicting developmental outcomes demands a quantitative understanding of the flow of 39 information along the central dogma: how input transcription factors dictate the output rate of 40 1 of 69 Manuscript submitted to bioRxiv mRNA production, how this rate of mRNA production dictates cytoplasmic patterns of mRNA, 41 and how these mRNA patterns lead to protein patterns that feed back into the gene regulatory 42 network. While the connection between transcription factor concentration and output mRNA 43 production rate has been the subject of active research over the last three decades (Lawrence 44 et al.connection between this output rate and 47 In order to uncover the quantitative contribution of these three regulatory strategies to pattern 62 formation, and to determine whether other regulatory strategies are at play, it is necessary to 63 measure the rate of RNA polymerase loading in individual nuclei, in real time, in a living embryo. 64 2 of 69 Manuscript submitted to bioRxiv However, to date, most studies have relied on fixed-tissue techniques such as mRNA FISH and 65 immunofluorescence in order to obtain snapshots of the cytoplasmic distributions of mRNA and 66 protein as development progresses (Jaeger et al., 2004; Fakhouri et al., 2010; Parker et al., 2011; 67 Estrada et al., 2016; Crocker et al., 2016; Verd et al., 2017; Park et al., 2018). Such techniques 68 are virtually silent regarding the regulation of single-cell gene expression over time, and are thus 69 ill-suited to the study of how spatiotemporal variations in transcriptional dynamics give rise to 70 patterns of cytoplasmic mRNA. 71...

Enhancer priming enables fast and sustained transcriptional responses to Notch signaling

“…Calculation of the autocorrelation function in traces from these nuclei suggest very slow transcriptional dynamics ( Fig. S4ED) (Desponds et al 2016;Lammers et al 2018), which would be consistent with one long period of activity as opposed to overlapping short bursts. This fits with a model where promoters can exist in a permissive active state, during which many "convoys" of polymerase can be fired without the promoter reverting to a fully inactive condition (Tantale et al 2016).…”

“…4E). In most previous enhancers analyzed in this way, the off period is the most affected, leading to changes in the bursting frequency (Fukaya et al 2016;Fritzsch et al 2018;Lammers et al 2018). However, when we quantified the effect from different doses of NICD on sim in the ME we found that the bursting length consistently increased with higher amounts of NICD whereas the off period between bursts remained constant ( Fig.…”

“…Changes in the duration or frequency of the bursts lead to an overall increase in transcription. Most commonly, differences in enhancer activity have been attributed to changes in the switching on probability (Kon), leading to changes in burst frequency (Larson et al 2013;Senecal et al 2014;Fukaya et al 2016;Fritzsch et al 2018;Lammers et al 2018;Berrocal et al 2018). We were therefore surprised to find that higher doses of NICD did not increase burst frequency.…”

“…Rarely have transcription dynamics been analyzed in vivo in the real-time of developmental signalling pathways, so we know little about how recipient enhancers decipher the signals. For example, enhancers could respond in a digital manner, working as simple on-off switches, or as analog devices, operating as a rheostat so that signal levels can modulate the output (Blackwood and Kadonaga 1998;Garcia et al 2013;Lammers et al 2018). In either case they must also have the capability to detect and transduce key parameters to the transcription machinery, such as signal duration and thresholds.…”

“…Since forced looping of the beta-globin enhancer to its promoter led to an increase in burst frequency (Bartman et al 2016), it has been proposed that transcription factors activate transcription by modulating enhancer-promoter interactions, and hence bursting frequency; although other studies suggest enhancer-promoter interactions are not the underlying basis of transcriptional bursting (Lim et al 2018;Chen et al 2018). Though the molecular origin of bursting remains unknown, bursting frequency rather than burst duration or amplitudes seems to be the major parameter modulated in different species and contexts (So et al 2011;Senecal et al 2014;Xu et al 2015;Desponds et al 2016;Padovan-Merhar et al 2015;Lammers et al 2018;Berrocal et al 2018). For example, enhancers controlling early patterning genes in Drosophila embryos produce similar bursting size but have different bursting frequencies, which can be attenuated by the presence of insulators (Fukaya et al 2016).…”