Lewis Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Cell Rep. 2022 Mar 22;38(12):110543. doi: 10.1016/j.celrep.2022.110543.
Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal the kinetics of gene expression downstream of a developmental transcription factor in vivo. We engineer light-controlled versions of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Krüppel. In addition, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a noncanonical role for Bicoid in directly suppressing knirps transcription. Acute modulation of transcription factor concentration while recording output gene activity represents a powerful approach for studying developmental gene networks in vivo.
发育模式网络受多种输入和反馈连接调控，这些连接迅速重塑基因表达，限制了仅从缓慢遗传扰动中获得的信息。在这里，我们展示了快速光遗传学刺激、实时转录报告器和简化的遗传背景可以结合使用，以揭示体内发育转录因子下游基因表达的动力学。我们设计了光控版本的 Bicoid 转录因子，并研究了它们对胚胎中下游间隙基因的影响。我们的结果再现了已知的关系，包括快速的 Bicoid 依赖性转录的 giant 和 hunchback，以及对 Krüppel 的延迟抑制。此外，我们发现 knirps 的后模式表现出对 Bicoid 扰动的快速但反转的反应，这表明 Bicoid 在直接抑制 knirps 转录中具有非典型作用。在记录输出基因活性的同时急性调节转录因子浓度，代表了研究体内发育基因网络的一种强大方法。
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