Precise temporal control of neuroblast migration through combined regulation and feedback of a Wnt receptor
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eLife assessment
This paper deals with an important unsolved problem in developmental biology of how cells execute their dynamics at the right time. The study combines compelling quantitative single cell and single transcript experiments with genetic perturbations and computational modelling and provides important insights into how the timing of transcription is regulated. The work would be strengthened by better integration of modeling and data.
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Abstract
Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that controls neuroblast migration in Caenorhabditis elegans as a tractable system to study a robust, cell-intrinsic timing mechanism in vivo. Single-molecule mRNA quantification showed that the expression of the receptor increases non-linearly, a dynamic that is predicted to enhance timing precision over an unregulated, linear increase in timekeeper abundance. We show that this upregulation depends on transcriptional activation, providing in vivo evidence for a model in which the timing of receptor expression is regulated through an accumulating activator that triggers expression when a specific threshold is reached. This timing mechanism acts across a cell division that occurs in the neuroblast lineage and is influenced by the asymmetry of the division. Finally, we show that positive feedback of receptor expression through the canonical Wnt pathway enhances temporal precision. We conclude that robust cell-intrinsic timing can be achieved by combining regulation and feedback of the timekeeper gene.
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eLife assessment
This paper deals with an important unsolved problem in developmental biology of how cells execute their dynamics at the right time. The study combines compelling quantitative single cell and single transcript experiments with genetic perturbations and computational modelling and provides important insights into how the timing of transcription is regulated. The work would be strengthened by better integration of modeling and data.
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Reviewer #1 (Public Review):
The authors study the control of the timing of Q neuroblast migration, through the precisely timed expression of the Wnt receptor MIG-1/Frizzled, which halts migration of the QR.pa cell at its intended position. Understanding the underlying mechanism is important, as similar mechanisms might play a role in controlling the timing of biological processes in development much more broadly. The authors use precise measurements of mig-1 mRNA molecules, fitted to mathematical models of different mechanisms to control the timing of mig-1 expression, and couple this with experimental perturbations of mig-1 expression. In this way, the authors convincingly show that mig-1 dynamics is best explained by a model where mig-1 expression is controlled by the accumulation of an activator, rather than the degradation of a …
Reviewer #1 (Public Review):
The authors study the control of the timing of Q neuroblast migration, through the precisely timed expression of the Wnt receptor MIG-1/Frizzled, which halts migration of the QR.pa cell at its intended position. Understanding the underlying mechanism is important, as similar mechanisms might play a role in controlling the timing of biological processes in development much more broadly. The authors use precise measurements of mig-1 mRNA molecules, fitted to mathematical models of different mechanisms to control the timing of mig-1 expression, and couple this with experimental perturbations of mig-1 expression. In this way, the authors convincingly show that mig-1 dynamics is best explained by a model where mig-1 expression is controlled by the accumulation of an activator, rather than the degradation of a repressor, which is an important result. In addition, they show that the asymmetric division of QR.p into the larger QR.pa and smaller QR.pp cells is important for proper mig-1 expression in Qr.pa, likely by asymmetric inheritance of the activator. In the process, the authors identify novel conserved binding motifs that are responsible for different aspects of mig-1 dynamics, which will potentially allow identifying the putative activator in the future.
In its current form, I find the manuscript has two main weak points: First, the connection between the experiments and models is relatively weak. Now, the model is mostly used to aid the interpretation of experiments, by predicting rough trends. However, even though the model is in principle fitted to the experimental data in some cases, a detailed comparison between experimental results and the model is often lacking. For example, there are multiple occasions where the data appears to not fit the model in some aspects, but the potential origin of these mismatches is typically not discussed. Second, the authors present experimental evidence of an earlier model prediction, that positive feedback loops in mig-1 expression reduce variability in timing. Here, the authors speculate that this feedback loop might be due to the activation of mig-1 expression by mig-1-induced Wnt signaling, which in itself is an interesting idea. However, the genetic perturbation used here - manipulation of the Wnt pathway, rather than perturbing specifically the induction of mig-1 expression by Wnt signaling - likely changes the expression of many genes in the cell, making it difficult to establish whether the increased variability in Qr.pa position is indeed due breaking the proposed feedback loop.
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Reviewer #2 (Public Review):
Schild et al. investigate the regulation of temporal control during neuroblast migration in the roundworm C. elegans. The authors find that expression of the Wnt pathway receptor Mig1 is regulated early through a specific noncoding conserved intronic element and later through two specific upstream conserved DNA elements. The expression levels of Mig1 in QR.pa cells are further regulated through Ced-3 and pig-1. The variability in the timing of later expression of Mig1 in QR.pa cells through bar-1 or a terminally truncated version of Bar1 was modulated but the mean expression did not change.
The single molecule RNA-FISH data is strong, and this method is sensitive enough to detect differences between different single cells and mutants. The mutants are very precise and straightforward to interpret. An …
Reviewer #2 (Public Review):
Schild et al. investigate the regulation of temporal control during neuroblast migration in the roundworm C. elegans. The authors find that expression of the Wnt pathway receptor Mig1 is regulated early through a specific noncoding conserved intronic element and later through two specific upstream conserved DNA elements. The expression levels of Mig1 in QR.pa cells are further regulated through Ced-3 and pig-1. The variability in the timing of later expression of Mig1 in QR.pa cells through bar-1 or a terminally truncated version of Bar1 was modulated but the mean expression did not change.
The single molecule RNA-FISH data is strong, and this method is sensitive enough to detect differences between different single cells and mutants. The mutants are very precise and straightforward to interpret. An additional strength is that many cells and replicas have been measured. The data analysis is simple.
The proposed model is simple with few intuitive parameters. This makes parameter identification straightforward. The qualitative predictions do make sense and are consistent with most experimental observations.
Overall the manuscript addresses the important question of timing regulation in transcription.
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