A timer gene network is spatially regulated by the terminal system in the Drosophila embryo
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Evaluation Summary:
Through the use of multiplexed in situ hybridization with careful embryo staging, this manuscript represents exemplary documentation of dynamic gene expression patterns in early fly development. By comparison of these patterns in various mutant combinations, a simple logical model for specification of expression is proposed. This manuscript will be of broad significance to developmental biologists interested in embryo segmentation and gene regulatory networks underpinning patterning.
(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. Reviewer #1 agreed to share their name with the authors.)
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Abstract
In insect embryos, anteroposterior patterning is coordinated by the sequential expression of the ‘timer’ genes caudal , Dichaete, and odd-paired , whose expression dynamics correlate with the mode of segmentation. In Drosophila , the timer genes are expressed broadly across much of the blastoderm, which segments simultaneously, but their expression is delayed in a small ‘tail’ region, just anterior to the hindgut, which segments during germband extension. Specification of the tail and the hindgut depends on the terminal gap gene tailless , but beyond this the regulation of the timer genes is poorly understood. We used a combination of multiplexed imaging, mutant analysis, and gene network modelling to resolve the regulation of the timer genes, identifying 11 new regulatory interactions and clarifying the mechanism of posterior terminal patterning. We propose that a dynamic Tailless expression gradient modulates the intrinsic dynamics of a timer gene cross-regulatory module, delineating the tail region and delaying its developmental maturation.
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Evaluation Summary:
Through the use of multiplexed in situ hybridization with careful embryo staging, this manuscript represents exemplary documentation of dynamic gene expression patterns in early fly development. By comparison of these patterns in various mutant combinations, a simple logical model for specification of expression is proposed. This manuscript will be of broad significance to developmental biologists interested in embryo segmentation and gene regulatory networks underpinning patterning.
(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. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Clark, Battistara, and Benton investigated the formation of the posterior terminal segments of the Drosophila embryo using carefully staged multiplexed Hybridization Chain Reaction (HCR), quantitative imaging, genetics, and computational modeling. Through the HCR studies, Clark et al. provide some of the most detailed and comprehensive documentation of the dynamic gene expression patterns governing the formation of the posterior terminal region. Through this work, they define two additional parasegmental boundaries that form in this posterior region following the establishment of the fourteen parasegments typically ascribed to the fly embryo. They demonstrate a role for a cross-regulatory 'timer gene network' comprised of Odd-paired, Caudal, and Dichaete to specify this posterior segmental unit. By comparing …
Reviewer #1 (Public Review):
Clark, Battistara, and Benton investigated the formation of the posterior terminal segments of the Drosophila embryo using carefully staged multiplexed Hybridization Chain Reaction (HCR), quantitative imaging, genetics, and computational modeling. Through the HCR studies, Clark et al. provide some of the most detailed and comprehensive documentation of the dynamic gene expression patterns governing the formation of the posterior terminal region. Through this work, they define two additional parasegmental boundaries that form in this posterior region following the establishment of the fourteen parasegments typically ascribed to the fly embryo. They demonstrate a role for a cross-regulatory 'timer gene network' comprised of Odd-paired, Caudal, and Dichaete to specify this posterior segmental unit. By comparing expression patterns of the timer network in an array of mutants defective for terminal specification and patterning, they address a longstanding question of how the specification of the terminus of the embryo by maternal Torso receptor tyrosine kinase signaling coordinates with the segmentation of the embryo "trunk". These conclusions are rigorously supported by excellent imaging and staging of samples, as well as quantification. Based on these observations, a simple computational model is proposed that integrates observed gene expression states to explain the formation of the interface between the segmental primordium and the embryonic terminus.
Strengths:
The data collection for this manuscript is rigorous, comprehensive, and excellent. As in prior publications from the lead author, the images collected here will be the standard documents for dynamic expression patterns of Drosophila segmentation/patterning genes.The logic behind the experiments is clearly described and performed comprehensively by exhaustively staining for multiple panels of markers in all relevant mutant backgrounds. The scholarship operating behind the scenes is likewise excellent. Several experiments, performed authoritatively here, clarify generalizations or ambiguities in the literature from the past thirty years on this subject.
The central question addressed in the manuscript is a longstanding unanswered question about a developmental model system that has been exhaustively studied over four decades. The concluding proposed gene regulatory network is well supported and opens up several lines of possible future inquiry in both Drosophila as well as other insect species.
Weaknesses:
The manuscript often drills down deep on details that will be of interest to only the most dedicated Drosophilists, which is appreciated, but also significantly derails and dilutes the central question of the manuscript at times. A more streamlined presentation, adjustments to the presentation of figures, and perhaps (greater) use of appendices for a more nuanced presentation of results would improve readability and open the results to a broader readership. -
Reviewer #2 (Public Review):
Clark et al use multiplex fluorescent in situ hybridisation and confocal imaging of wild-type and mutant Drosophila embryos to characterise the relationship between timer genes and posterior terminal genes during tail patterning. The combination of exonic and intronic probes, along with antibody staining, is powerful in defining the relationship between expression patterns. The findings are used to generate a regulatory network and a logical computational model, with complete justification for some of the positive/negative interactions defined as explained in Table 1. Overall, the authors show that, in contrast to the simultaneous segmentation of the bulk of the segments in the Drosophila embryo, two parasegment-like boundaries are patterned sequentially from the terminal region during gastrulation.
In …
Reviewer #2 (Public Review):
Clark et al use multiplex fluorescent in situ hybridisation and confocal imaging of wild-type and mutant Drosophila embryos to characterise the relationship between timer genes and posterior terminal genes during tail patterning. The combination of exonic and intronic probes, along with antibody staining, is powerful in defining the relationship between expression patterns. The findings are used to generate a regulatory network and a logical computational model, with complete justification for some of the positive/negative interactions defined as explained in Table 1. Overall, the authors show that, in contrast to the simultaneous segmentation of the bulk of the segments in the Drosophila embryo, two parasegment-like boundaries are patterned sequentially from the terminal region during gastrulation.
In general, the data are high quality and the conclusions are supported by the data.
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Reviewer #3 (Public Review):
In this manuscript, the authors investigate the regulation of the 'timer' genes, cad, D, and opa, and their roles in the formation of the most posterior segments of Drosophila using analysis of gene expression genetics and modelling.
The authors re-examined posterior expression of en and wg and corroborated previous findings of additional stripes corresponding to the most posterior parasegments. This expression is pre-figured by slp and eve expression. This evidences sequential addition of the terminal segments and segment polarity expression of eve that is not prefigured by a pair-rule expression.
Analysis of the three timer genes' expression in the posterior region shows that they are expressed in the same temporal sequence as in the trunk.
The authors then showed that there is cross-regulation of timer …
Reviewer #3 (Public Review):
In this manuscript, the authors investigate the regulation of the 'timer' genes, cad, D, and opa, and their roles in the formation of the most posterior segments of Drosophila using analysis of gene expression genetics and modelling.
The authors re-examined posterior expression of en and wg and corroborated previous findings of additional stripes corresponding to the most posterior parasegments. This expression is pre-figured by slp and eve expression. This evidences sequential addition of the terminal segments and segment polarity expression of eve that is not prefigured by a pair-rule expression.
Analysis of the three timer genes' expression in the posterior region shows that they are expressed in the same temporal sequence as in the trunk.
The authors then showed that there is cross-regulation of timer genes by examining the effect of expression of these genes in mutants of the others. The boundaries of these genes in the posterior of the embryo also appear to be regulated by tll and hkb, and the posterior patterning of these genes is lost in tor mutants.
Taken together the gene expression and genetic analyses allow the authors to 'wire' the timer genes into the GRN for posterior patterning significantly adding to our understanding of these interactions as summarised in fig 8.
Finally, the authors modelled these interactions. They found that simulating the inferred genetic interactions was broadly able to explain their observations and help to better understand the patterning of the posterior of the embryo.
This is very solid work, illustrated by excellent figures, that provides new insights into the regulation of posterior development in Drosophila and has important implications for the regulation of segmentation in other insects.
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