Delilah, prospero, and D-Pax2 constitute a gene regulatory network essential for the development of functional proprioceptors
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Evaluation Summary:
The study is carefully performed and provides compelling evidence for a gene network involved in generating different sensory cell types from a common progenitor, showing how an enhancer can integrate a gene regulatory network and guide a cell-fate decision.
(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 #2 agreed to share their name with the authors.)
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Abstract
Coordinated animal locomotion depends on the development of functional proprioceptors. While early cell-fate determination processes are well characterized, little is known about the terminal differentiation of cells within the proprioceptive lineage and the genetic networks that control them. In this work we describe a gene regulatory network consisting of three transcription factors–Prospero (Pros), D-Pax2, and Delilah (Dei)–that dictates two alternative differentiation programs within the proprioceptive lineage in Drosophila . We show that D-Pax2 and Pros control the differentiation of cap versus scolopale cells in the chordotonal organ lineage by, respectively, activating and repressing the transcription of dei . Normally, D-Pax2 activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. We further show that D-Pax2 and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer in which two D-Pax2- and three Pros-binding sites were identified experimentally. When this enhancer was removed from the fly genome, the cap- and ligament-specific expression of dei was lost, resulting in loss of chordotonal organ functionality and defective larval locomotion. Thus, coordinated larval locomotion depends on the activity of a dei enhancer that integrates both activating and repressive inputs for the generation of a functional proprioceptive organ.
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Evaluation Summary:
The study is carefully performed and provides compelling evidence for a gene network involved in generating different sensory cell types from a common progenitor, showing how an enhancer can integrate a gene regulatory network and guide a cell-fate decision.
(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 #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This work by Avetisyan et al. is focused on uncovering the developmental processes underlying cell type differentiation events during later stages of larval proprioceptive chordotonal organs (ChO) in Drosophila. Making use of an enhancer recently identified by this research group, and building on a previous RNAi screen, the authors find that two transcription factors known to regulate sensory organ development in other contexts - D-Pax2 and Prospero (pros) - function antagonistically to regulate the expression the expression of another transcription factor, Delilah (dei), in two "cousin" cell types in the LCh5 cluster of ChOs. Studies include standard loss- and gain-of-function experiments, gel shift assays, and in vivo reporter analysis to show that Pax2 promotes dei expression in PIIa-derived cap cells, …
Reviewer #1 (Public Review):
This work by Avetisyan et al. is focused on uncovering the developmental processes underlying cell type differentiation events during later stages of larval proprioceptive chordotonal organs (ChO) in Drosophila. Making use of an enhancer recently identified by this research group, and building on a previous RNAi screen, the authors find that two transcription factors known to regulate sensory organ development in other contexts - D-Pax2 and Prospero (pros) - function antagonistically to regulate the expression the expression of another transcription factor, Delilah (dei), in two "cousin" cell types in the LCh5 cluster of ChOs. Studies include standard loss- and gain-of-function experiments, gel shift assays, and in vivo reporter analysis to show that Pax2 promotes dei expression in PIIa-derived cap cells, whereas pros suppresses this activation in the PIIb-derived scolopale cell through experimentally-defined binding sites. CRISPR-mediated deletion of the enhancer confirms that the enhancer is sufficient to drive expression of dei in ChOs, with its deletion leading to defects in larval locomotion.
Overall, the study is carefully performed and the data generally support the model that Pax2 and Pros function antagonistically to control dei expression during ChO development. Quantifications of the expression levels of site-directed mutants, and of the larval locomotion were also valuable for assessing the specific contributions of particularly cis-regulatory sequences. Appropriate discussion of previous findings in the fly eye and mechanosensory system demonstrating a similar genetic relationship between Pax2 and Pros is included. The authors also recognize that the precise mechanism by which this antagonism is achieved remains unanswered, despite their identification of overlapping binding sites within the minimal dei ChO enhancer. Together, this study helps to clarify downstream roles for Pros and Pax2 in chordotonal organ formation, and further establishes that the antagonistic relationship between these factors is broadly required during sensory system development in Drosophila.
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Reviewer #2 (Public Review):
In this manuscript, the authors have performed a careful analysis of an enhancer that they have identified within the gene, Dei, in the context of sense organ development in larval Drosophila. The study is interesting and important because the authors demonstrate that a 262bp enhancer of Dei contains binding sites for two transcription factors, sv and pros. They show that sv binds this enhancer to activate Dei expression in some cells of the sense organ, and pros binds it to repress Dei expression in other cells. Thus, this 262 bp region integrates both activation and repression of Dei, ensuring the appropriate expression of this gene and therefore appropriate cell fate decisions within the sense organ lineage. Most interestingly, the authors demonstrate this via a combination of methods, including …
Reviewer #2 (Public Review):
In this manuscript, the authors have performed a careful analysis of an enhancer that they have identified within the gene, Dei, in the context of sense organ development in larval Drosophila. The study is interesting and important because the authors demonstrate that a 262bp enhancer of Dei contains binding sites for two transcription factors, sv and pros. They show that sv binds this enhancer to activate Dei expression in some cells of the sense organ, and pros binds it to repress Dei expression in other cells. Thus, this 262 bp region integrates both activation and repression of Dei, ensuring the appropriate expression of this gene and therefore appropriate cell fate decisions within the sense organ lineage. Most interestingly, the authors demonstrate this via a combination of methods, including generating mutants of the enhancer that phenocopies the gene mutant in cell-fate specification as well as behaviour.
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Reviewer #3 (Public Review):
In this paper, Avetisyan et al use Drosophila genetics, transgenic reporter assays, in vitro DNA binding assays, genome editing, and larval movement assays to study how the Pros and D-Pax2 transcription factors differentially impact the expression of delilah (dei) and ultimately, the function of the Drosophila abdominal lateral chordotonal organs (lch5). A prior RNAi screen revealed that both Pros and sv (D-Pax2) were required for proper dei expression in the lch5 organ. Building off previous publications, the authors focus on a specific enhancer within an intron of dei that is sufficient to mediate expression in cap and attachment cells (but not scolopale cells) in the lch5 organ. The authors recapitulated the RNAi findings using loss-of-function alleles for sv and Pros, and the authors used …
Reviewer #3 (Public Review):
In this paper, Avetisyan et al use Drosophila genetics, transgenic reporter assays, in vitro DNA binding assays, genome editing, and larval movement assays to study how the Pros and D-Pax2 transcription factors differentially impact the expression of delilah (dei) and ultimately, the function of the Drosophila abdominal lateral chordotonal organs (lch5). A prior RNAi screen revealed that both Pros and sv (D-Pax2) were required for proper dei expression in the lch5 organ. Building off previous publications, the authors focus on a specific enhancer within an intron of dei that is sufficient to mediate expression in cap and attachment cells (but not scolopale cells) in the lch5 organ. The authors recapitulated the RNAi findings using loss-of-function alleles for sv and Pros, and the authors used gain-of-function assays to further show that these two factors impact dei expression in lch5 organs. A yeast 1-hybrid assay was used to identify D-Pax2 as a direct binding factor to the dei enhancer and EMSAs were used to map D-Pax2 and Pros binding sites, which were found to regulate dei enhancer activity in transgenic reporter assays. Lastly, the authors used genome editing to specifically remove this dei enhancer and found that its loss resulted in abnormal larvae movement.
Overall, the experiments are largely supportive of the main authors conclusions that Pros and D-Pax2 directly regulate dei expression in distinct cell types of the lch5 organ. However, I do have several experimental concerns, which are raised below, that would need to be addressed to further strengthen this conclusion. Moreover, while the finding that D-Pax2 activates and Pros represses dei presents a compelling gene regulatory module, the overall advance is rather modest as these results focus only on a single enhancer in a single gene and do not significantly describe how the differential regulation of dei leads to distinct gene regulatory networks that define scolopale vs attachment/ligament cell fate. In addition, the authors only explore the D-Pax2/Pros/dei relationship within one type of proprioceptor (lch5) that is specified in the Drosophila embryo and functions within the larva, and not within other proprioceptor sensory organs of the embryo (dch3/vchA/B, where I assume it is similar) and not in sensory organs that are also specified by atonal later in the larva (i.e. the leg proprioceptors, Johnston's organs, etc) or in the external sensory organs of the wing (where dei is also expressed). Without such findings, it is unclear if this antagonistic relationship between Prox/dPax2 is broadly used to specify different fates in many different sensory organs.
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