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Dynamics of hunchback translation in real time and at single mRNA resolution in the Drosophila embryo.

ABSTRACT: The Hunchback (Hb) transcription factor is critical for anterior-posterior patterning of the Drosophila embryo. Despite the maternal hb mRNA acting as a paradigm for translational regulation, due to its repression in the posterior of the embryo, little is known about the translatability of zygotically transcribed hb mRNAs. Here we adapt the SunTag system, developed for imaging translation at single mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using single-molecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. While the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.

SUBMITTER: Vinter DJ

PROVIDER: S-EPMC8077512 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:During embryonic development, the positional information provided by concentration gradients of maternal factors directs pattern formation by providing spatially dependent cues for gene expression. In the fruit fly, Drosophila melanogaster, a classic example of this is the sharp on-off activation of the hunchback (hb) gene at midembryo, in response to local concentrations of the smooth anterior-posterior Bicoid (Bcd) gradient. The regulatory region for hb contains multiple binding sites for the Bcd protein as well as multiple binding sites for the Hb protein. Some previous studies have suggested that Bcd is sufficient for properly sharpened Hb expression, yet other evidence suggests a need for additional regulation. We experimentally quantified the dynamics of hb gene expression in flies that were wild-type, were mutant for hb self-regulation or Bcd binding, or contained an artificial promoter construct consisting of six Bcd and two Hb sites. In addition to these experiments, we developed a reaction-diffusion model of hb transcription, with Bcd cooperative binding and hb self-regulation, and used Zero Eigenvalue Analysis to look for multiple stationary states in the reaction network. Our model reproduces the hb developmental dynamics and correctly predicts the mutant patterns. Analysis of our model indicates that the Hb sharpness can be produced by spatial bistability, in which hb self-regulation produces two stable levels of expression. In the absence of self-regulation, the bistable behavior vanishes and Hb sharpness is disrupted. Bcd cooperative binding affects the position where bistability occurs but is not itself sufficient for a sharp Hb pattern. Our results show that the control of Hb sharpness and positioning, by hb self-regulation and Bcd cooperativity, respectively, are separate processes that can be altered independently. Our model, which matches the changes in Hb position and sharpness observed in different experiments, provides a theoretical framework for understanding the data and in particular indicates that spatial bistability can play a central role in threshold-dependent reading mechanisms of positional information.

Project description:In early development, genes are expressed in spatial patterns which later define cellular identities and tissue locations. The mechanisms of such pattern formation have been studied extensively in early Drosophila (fruit fly) embryos. The gap gene hunchback (hb) is one of the earliest genes to be expressed in anterior-posterior (AP) body segmentation. As a transcriptional regulator for a number of downstream genes, the spatial precision of hb expression can have significant effects in the development of the body plan. To investigate the factors contributing to hb precision, we used fine spatial and temporal resolution data to develop a quantitative model for the regulation of hb expression in the mid-embryo. In particular, modelling hb pattern refinement in mid nuclear cleavage cycle 14 (NC14) reveals some of the regulatory contributions of simultaneously-expressed gap genes. Matching the model to recent data from wild-type (WT) embryos and mutants of the gap gene Krüppel (Kr) indicates that a mid-embryo Hb concentration peak important in thoracic development (at parasegment 4, PS4) is regulated in a dual manner by Kr, with low Kr concentration activating hb and high Kr concentration repressing hb. The processes of gene expression (transcription, translation, transport) are intrinsically random. We used stochastic simulations to characterize the noise generated in hb expression. We find that Kr regulation can limit the positional variability of the Hb mid-embryo border. This has been recently corroborated in experimental comparisons of WT and Kr- mutant embryos. Further, Kr regulation can decrease uncertainty in mid-embryo hb expression (i.e. contribute to a smooth Hb boundary) and decrease between-copy transcriptional variability within nuclei. Since many tissue boundaries are first established by interactions between neighbouring gene expression domains, these properties of Hb-Kr dynamics to diminish the effects of intrinsic expression noise may represent a general mechanism contributing to robustness in early development.

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Dynamics of hunchback translation in real time and at single mRNA resolution in the Drosophila embryo.

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