Generation and timing of graded responses to morphogen gradients
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Evaluation Summary:
This is an interesting study addressing an overlooked feature of morphogen gradient interpretation. By studying transcriptional activation of early zygotic genes during early Drosophila development the authors' findings suggest that morphogens, which are known to pattern tissues in distinct domain of gene expression, can also induce a graded transcriptional response within these domains.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.
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Evaluation Summary:
This is an interesting study addressing an overlooked feature of morphogen gradient interpretation. By studying transcriptional activation of early zygotic genes during early Drosophila development the authors' findings suggest that morphogens, which are known to pattern tissues in distinct domain of gene expression, can also induce a graded transcriptional response within these domains.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors use smFISH to study the activation dynamics of the ventrally-expressed T48 gene. They observe that different concentrations of Dorsal (in a gradient with peak levels in the ventral-most nuclei) lead to 1) different probabilities of "priming" and thus timing of activation, and 2) RNA polymerase loading. Together these define a dual mechanism of T48 mRNA accumulation in a graded manner. The experiments are elegant and the data well presented. The issue I have is that the idea of graded activation by Dorsal is not new. It is known that Dl and Twi regulate T48 (as hypothesized on lines 204 and 214), and that the T48 enhancer contains both TF binding sites, as this was published by the Lim lab in PNAS - Keller et al, 2020. This paper reported on the dynamics of transcriptional activation of T48, using …
Reviewer #1 (Public Review):
The authors use smFISH to study the activation dynamics of the ventrally-expressed T48 gene. They observe that different concentrations of Dorsal (in a gradient with peak levels in the ventral-most nuclei) lead to 1) different probabilities of "priming" and thus timing of activation, and 2) RNA polymerase loading. Together these define a dual mechanism of T48 mRNA accumulation in a graded manner. The experiments are elegant and the data well presented. The issue I have is that the idea of graded activation by Dorsal is not new. It is known that Dl and Twi regulate T48 (as hypothesized on lines 204 and 214), and that the T48 enhancer contains both TF binding sites, as this was published by the Lim lab in PNAS - Keller et al, 2020. This paper reported on the dynamics of transcriptional activation of T48, using live imaging to examine the effects of Dorsal (by removing binding sites) on transcriptional activation timing and transcriptional output (similar to priming and Pol II loading rate in this study). The Keller et al. paper concluded that T48 "exhibits a dynamic expression pattern, where nuclei along the ventral midline are the first to begin transcription, with more lateral nuclei becoming active as NC14 progresses." It further showed that "Dl site modulations change spatial boundaries of t48, mostly by affecting the timing of activation and bursting frequency rather than transcriptional amplitude or bursting duration." Thus, the Keller et al. paper came to basically the same conclusion about the effects of Dl on T48 transcription. Even the experiment utilizing constitutively-active Dl had a similar effect on T48 as the Lim experiment optimizing Dl binding sites in the T48 enhancer.
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Reviewer #2 (Public Review):
The manuscript by Carmon et al investigates the mechanisms underlying the graded response to a morphogen gradient. Specifically they monitor the transcriptional response of two Dorsal targets, T48 and mist during mesoderm formation in Drosophila early embryos. For this, they employ single molecule FISH (smFISH) on wild type and ventralized embryos at various timing of mesoderm induction.
The major finding of this manuscript is that the gradual response of T48/mist relies on two complementary mechanisms:- the priming of these genes by Dorsal
- the loading rate of Pol II, also dependent on Dorsal nuclear levels.
The data are of high quality and represent solid quantitative information.
A weakness is that I find is the lack of functional analysis to validate some of the conclusions made from the quantitative …
Reviewer #2 (Public Review):
The manuscript by Carmon et al investigates the mechanisms underlying the graded response to a morphogen gradient. Specifically they monitor the transcriptional response of two Dorsal targets, T48 and mist during mesoderm formation in Drosophila early embryos. For this, they employ single molecule FISH (smFISH) on wild type and ventralized embryos at various timing of mesoderm induction.
The major finding of this manuscript is that the gradual response of T48/mist relies on two complementary mechanisms:- the priming of these genes by Dorsal
- the loading rate of Pol II, also dependent on Dorsal nuclear levels.
The data are of high quality and represent solid quantitative information.
A weakness is that I find is the lack of functional analysis to validate some of the conclusions made from the quantitative analysis of the transcriptional response.
For example, it would be exciting to assess the functional consequences of an intronless T48 gene by CRISPR gene editing. Phenotypes in terms of timing/coordination in the constriction of the actomyosin network would greatly improve the biological significance of the transcriptional findings of this manuscript.The overall conclusions of this paper are interesting and supported by their data.
Specific comments:
1-The word 'priming' is used in various instances (text and title of figures) but might be an over-interpretation. The results show the timing of transcriptional activation in various domains. A gradual activation could be interpreted as a gradual priming by a TF, but this is an interpretation.
In particular, while the pioneering action of Zelda has been demonstrated and provides its 'priming' capacity, a similar scenario has not yet been shown for Dorsal. Thus, priming should be discussed with respect to a coordinated action between Dorsal and Zelda. Alternatively, if the authors believe that it's an action solely based on Dorsal, then this should be demonstrated (e.g. RNAi Zelda) or at least discussed.By the way, manipulation of Dorsal binding sites or Zelda binding site at T48 mesodermal enhancer has already been performed (albeit in a transgenic context). The authors should discuss and cite this important work: Keller et al 2021, DOI: 10.1073/pnas.1917040117 .
The differential response to the Dorsal gradient shown for twi (switch like) compared to T48 (gradual) is an interesting paradigm. It would have been interesting to discuss the potential mechanisms supporting this differential response. For example, the contribution of Dorsal binding sites types and arrangement (grammar) and that of pioneering activity of Zelda. Many papers determined the contribution of Zelda binding to fostering the response to Dorsal gradient (with static approaches Crocker et al, 2017; Foo et al, Sun et al., or with live imaging Yamada et al. 2019, Dufourt et al. 2018). It would be interesting to discuss these results.
2-It is well known that expression is extremely dynamic during the course of nc14. The authors usually show two images, and state that one is an earlier stage. I could not find how embryonic staging was performed, with the exception of the statement line 205 'monitoring embryos of different ages (as defined by the number of T48 TSs)'.
A more quantitative assessment of embryonic timing by examining the level of membrane invagination for the embryo treated by smFISH and quantification would be preferable.
Moreover, it would be exciting to compare the quantifications of %TS activation from smFISH with those already published, from MS2-tagged lines (Lim et al. 2017). -
Reviewer #3 (Public Review):
This is an interesting study addressing an overlooked feature of morphogen gradient interpretation. By studying transcriptional activation of early zygotic genes during early Drosophila development the authors' findings suggest that morphogens, which are known to pattern tissues in distinct domain of gene expression, can also induce a graded transcriptional response within these domains. They speculate this might help orchestrating the spatiotemporal organization of downstream morphogenetic movements. While the results presented convincingly demonstrate graded activation of target genes, the underlying mechanisms remain unclear and additional information are needed to link the expression of these genes to the activity of Dorsal. Furthermore, whether the graded transcription of the genes analysed is …
Reviewer #3 (Public Review):
This is an interesting study addressing an overlooked feature of morphogen gradient interpretation. By studying transcriptional activation of early zygotic genes during early Drosophila development the authors' findings suggest that morphogens, which are known to pattern tissues in distinct domain of gene expression, can also induce a graded transcriptional response within these domains. They speculate this might help orchestrating the spatiotemporal organization of downstream morphogenetic movements. While the results presented convincingly demonstrate graded activation of target genes, the underlying mechanisms remain unclear and additional information are needed to link the expression of these genes to the activity of Dorsal. Furthermore, whether the graded transcription of the genes analysed is translated in corresponding gradients of protein activity and the impact on morphogenesis remain to be investigated.
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