Biology subjects

Crocker, J.

Publications and source records attributed to Crocker, J..

15 recordsLinked to original sources

bioRxiv2026

Interallelic cis-regulatory dominance promotes robustness and evolutionary innovation

Dominance is a central principle of genetics, yet the mechanistic basis and the evolutionary consequences of dominance arising from cis-regulatory variation remain poorly understood. We examined the evolutionary trajectories of a pleiotropic developmental enhancer in Drosophila. A genotype-phenotype map between D. melanogaster and D. simulans enhancer sequences reveals extensive epistasis, and many homozygous evolutionary paths reduce transcriptional output. In heterozygotes, however, regulatory dominance masks variants that reduce gene expression, potentially relaxing evolutionary constraints. Using allele-specific reporters and imaging, we show that this dominance arises from interallelic interactions (also known as transvection) reinforced by transcriptional hubs. Importantly, this enhancer dominance is cell-type specific, raising the possibility that it conceals deleterious effects in essential tissues while revealing novel, ectopic activity in others. Interallelic regulatory hubs may therefore expand the range of mutational paths available to diploid genomes while preserving essential transcriptional output.

evolutionary biology↗

bioRxiv2025

The evolution of mutation rates in the light of development and cell-lineage selection

Mutation rates drive the pace and potential of evolutionary change. However, to better understand the evolutionary implications of mutation rates, there is a need to uncover the causes of their diversification. In multicellular organisms, all mutations first arise in a single cell in a developmental context. Whether a mutation enters a populations gene pool can therefore depend on developmental events that affect the likelihood of mutant cell lineages of producing gametes. For this reason, the evolution of mutation rates in populations is governed not only by changes in the rates at which mutations occur at the molecular level, but also by changes in developmental features of organisms and how mutations impact cellular fitness during development. We present a theoretical framework that, supported by empirical data from mammals and experiments on the fruit fly, demonstrate how generational mutation rates can be shaped by changes in developmental parameters and intraorganismal selective processes even when molecular mutation rates are presumed constant. Our model highlights how the diversity of mutation rates observed across animals may be the byproduct of organismal development rather than the result of direct selection against mutator alleles. As such, development not only introduces phenotypic biases, it also shapes the rates and trajectories of genetic diversity and is thus at the core of evolutionary theory.

evolutionary biology↗

bioRxiv2024

Chromatin enables precise and scalable gene regulation with factors of limited specificity

Biophysical constraints limit the specificity with which transcription factors (TFs) can target regulatory DNA. While individual nontarget binding events may be low affinity, the sheer number of such interactions could present a challenge for gene regulation by degrading its precision or possibly leading to an erroneous induction state. Chromatin can prevent nontarget binding by rendering DNA physically inaccessible to TFs, at the cost of energy-consuming remodeling orchestrated by pioneer factors (PFs). Under what conditions and by how much can chromatin reduce regulatory errors on a global scale? We use a theoretical approach to compare two scenarios for gene regulation: one that relies on TF binding to free DNA alone, and one that uses a combination of TFs and chromatin-regulating PFs to achieve desired gene expression patterns. We find, first, that chromatin effectively silences groups of genes that should be simultaneously OFF, thereby allowing more accurate graded control of expression for the remaining ON genes. Second, chromatin buffers the deleterious consequences of nontarget binding as the number of OFF genes grows, permitting a substantial expansion in regulatory complexity. Third, chromatin-based regulation productively co-opts nontarget TF binding for ON genes in order to establish a "leaky" baseline expression level, which targeted activator or repressor binding subsequently up- or down-modulates. Thus, on a global scale, using chromatin simultaneously alleviates pressure for high specificity of regulatory interactions and enables an increase in genome size with minimal impact on global expression error. Significance StatementReliably keeping a gene off is as important as controlling its expression level when the gene is on. Yet both tasks become challenging in the packed nuclear environment of a eukaryotic cell, where the numerous and diverse regulatory proteins that are present cannot bind enhancers for target genes with perfect specificity. While regulatory schemes based on prokaryotic models would be overwhelmed by errors in such conditions, we show that chromatin-based regulation, an evolutionary innovation of eukaryotic cells, successfully rescues precise gene expression control by reliably keeping desired genes off. Our systems-level computational analysis demonstrates that this result is nontrivial, because chromatin opening must itself be correctly regulated. We furthermore identify when and how chromatin-based regulation outperforms alternative schemes.

biophysics↗

bioRxiv2024

LarvaTagger: Manual and automatic tagging of Drosophila larval behaviour

MotivationAs more behavioural assays are carried out in large-scale experiments on Drosophila larvae, the definitions of the archetypal actions of a larva are regularly refined. In addition, video recording and tracking technologies constantly evolve. Consequently, automatic tagging tools for Drosophila larval behaviour must be retrained to learn new representations from new data. However, existing tools cannot transfer knowledge from large amounts of previously accumulated data. We introduce LarvaTagger, a piece of software that combines a pre-trained deep neural network, providing a continuous latent representation of larva actions for stereotypical behaviour identification, with a graphical user interface to manually tag the behaviour and train new automatic taggers with the updated ground truth. ResultsWe reproduced results from an automatic tagger with high accuracy, and we demonstrated that pre-training on large databases accelerates the training of a new tagger, achieving similar prediction accuracy using less data. AvailabilityAll the code is free and open source. Docker images are also available. See git-lab.pasteur.fr/nyx/LarvaTagger.jl.

bioinformatics↗

bioRxiv2024

Pervasive sublethal effects of agrochemicals as contributing factors to insect decline

Insect biomass is declining across the globe at an alarming rate. Climate change and the widespread use of pesticides have been hypothesized as two underlying drivers. However, the lack of systematic experimental studies across chemicals and species limits our causal understanding of this problem. Here, we employed a chemical library encompassing 1024 different molecules--including insecticides, herbicides, fungicides, and plant growth inhibitors --to investigate how insect populations are affected by varying concentrations of pesticides, focusing on sublethal doses. Using a controlled laboratory pipeline for Drosophila melanogaster, we found that 57% of these chemicals affect the behavior of larvae at sublethal concentrations, and an even higher proportion compromises long-term survivability after acute exposure. Consistent with these results, we observed that exposure to chemicals at doses orders of magnitude below lethality induced widespread phosphorylation changes across the larval proteome. The effects of agrochemicals were amplified when the ambient temperature was increased by four degrees. We also tested the synergistic effects of multiple chemicals at doses found widely in nature and observed fitness-reducing changes in larval developmental time, behavior, and reproduction. Finally, we expanded our investigation to additional fly species, mosquitos, and butterflies and detected similar behavioral alterations triggered by pesticides at sublethal concentrations. Our results provide experimental evidence that strongly suggests sublethal doses of agrochemicals coupled with changes in environmental temperatures are contributing to the global decline in insect populations. We anticipate that our assays can contribute to improving chemical safety assessment, better protect the environment, secure food supplies, and safeguard animal and human health, as well as understand our rapidly changing world.

ecology↗

bioRxiv2022

Mutational scans reveal differential evolvability of Drosophila promoters and enhancers

Rapid enhancer and slow promoter evolution have been demonstrated through comparative genomics. However, it is not clear how this information is encoded genetically and if this can be used to place evolution in a predictive context. Part of the challenge is that our understanding of the potential for regulatory evolution is biased primarily toward natural variation or limited experimental perturbations. Here, to explore the evolutionary capacity of promoter variation, we surveyed an unbiased mutation library for three promoters in Drosophila melanogaster. We found that mutations in promoters had limited to no effect on spatial patterns of gene expression. Compared to developmental enhancers, promoters are more robust to mutations and have more access to mutations that can increase gene expression, suggesting that their low activity might be a result of selection. Consistent with these observations, increasing the promoter activity at the endogenous locus of shavenbaby led to increased transcription yet limited phenotypic changes. Taken together, developmental promoters may encode robust transcriptional outputs allowing evolvability through the integration of diverse developmental enhancers. Quote"Regulators, mount up [at transcriptional promoters]." - Warren G & Nate Dogg, 1994

evolutionary biology↗

bioRxiv2022

Laboratory evolution of flies to morphogen dosage via rapid maternal changes reveals predictable outcomes

Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges at a systems level remains limited. Here, we have examined the phenotypic consequences of disturbing the classic bicoid network in Drosophila, which is essential for anterior-posterior patterning in the early embryo. This network can be synthetically perturbed by increasing the dosage of bicoid, which causes a posterior shift of the networks regulatory outputs and a decrease in fitness. To directly monitor network changes across populations and time with extra copies of bicoid, we performed genome-wide EMS mutagenesis, followed by experimental evolution. After only 8-15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, we find that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. We extend our results to additional populations of flies, demonstrating predictability. Together, our results necessitate a broader view of regulatory network evolution at the systems level. In the future, such synthetic evolution approaches using animal models could provide a generalizable platform for studying the propagation of genetic perturbations across the many layers of complex multicellular systems.

developmental biology↗

bioRxiv2022

Enhancer architecture and chromatin accessibility constrain phenotypic space during development

Developmental enhancers are DNA sequences that when bound to transcription factors dictate specific patterns of gene expression during development. It has been proposed that the evolution of such cis-regulatory elements is a major source of adaptive evolution; however, the regulatory and evolutionary potential of such elements remains little understood, masked by selective constraints, drift and contingency. Here, using mutation libraries in Drosophila melanogaster embryos, we observed that most mutations in classical developmental enhancers led to changes in gene expression levels but rarely resulted in novel expression outside of the native cell- and tissue-types. In contrast, random sequences often acted as developmental enhancers, driving expression across a range of levels and cell-types, in patterns consistent with transcription factor motifs therein; random sequences including motifs for transcription factors with pioneer activity acted as enhancers even more frequently and resulting in higher levels of expression. Together, our findings suggest that the adaptive phenotypic landscapes of developmental enhancers are constrained by both enhancer architecture and chromatin accessibility. We propose that the evolution of existing enhancers is limited in its capacity to generate novel phenotypes, whereas the activity of de novo elements is a primary source of phenotypic novelty. QUOTE"Chance and chance alone has a message for us." Milan Kundera, The Unbearable Lightness of Being

developmental biology↗

bioRxiv2022

Developmental phenomics suggests that H3K4 monomethylation catalyzed by Trr functions as a phenotypic capacitor

How epigenetic modulators of gene regulation affect the development and evolution of animals has been difficult to ascertain. Despite the widespread presence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on animal development and viability. In this study, we performed quantitative, unbiased and multi-dimensional explorations of key phenotypes on Drosophila melanogaster with genetically induced hypomethylation. Hypomethylation reduced transcription factor enrichment in nuclear microenvironments, leading to reduced gene expression, and phenotypes outside of standard laboratory conditions. Our developmental phenomics survey further showed that H3K4me1 hypomethylation led to context-dependent changes in morphology, metabolism, and behavior. Therefore, H3K4me1 may contribute to phenotypic evolution as a phenotypic capacitor by buffering the effects of chance, genotypes and environmental conditions on transcriptional enhancers. Quote"Developmental biologists are often not so much opposed to a role for ecology as they simply ignore it" -Doug Erwin1

developmental biology↗

bioRxiv2021

Nuclear morphogenesis: forming a heterogeneous nucleus during embryogenesis

An embryo experiences progressively complex spatial and temporal patterns of gene expression that guide the morphogenesis of its body plan as it matures. Using super-resolution fluorescence microscopy in Drosophila melanogaster embryos, we observed a similar increase in complexity in the nucleus: the spatial distributions of transcription factors became increasingly heterogeneous as the embryo matured. We also observed a similar trend in chromatin conformation with the establishment of specific histone modification patterns. However, transcription sites of specific genes had distinct local preferences for histone marks separate from the average nuclear trend, depending on the time and location of their expression. These results suggest that reconfiguring the nuclear environment is an integral part of embryogenesis and that the physical organization of the nucleus is a key element in developmental gene regulation. Summary statementWe observed spatial rearrangements in the nucleus during embryo development, progressively forming a heterogeneous nuclear environment, paralleling the increasing complexity of the embryo body as morphogenesis progresses.

developmental biology↗

bioRxiv2020

Dense encoding of developmental regulatory information may constrain evolvability

Gene regulatory changes underlie much of phenotypic evolution. However, the evolutionary potential of regulatory evolution is unknown, because most evidence comes from either natural variation or limited experimental perturbations. Surveying an unbiased mutation library for a developmental enhancer in Drosophila melanogaster using an automated robotics pipeline, we found that most mutations alter gene expression. Our results suggest that regulatory information is distributed throughout most of a developmental enhancer and that parameters of gene expression--levels, location, and state--are convolved. The widespread pleiotropic effects of most mutations and the codependency of outputs may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse drosophilids reveal mainly stasis and apparent biases in the phenotypes influenced by this enhancer. Developmental enhancers may encode a much higher density of regulatory information than has been appreciated previously, which may impose constraints on regulatory evolution. Quote"Rock, robot rock Rock, robot rock Rock, robot rock" Daft Punk (2005)

evolutionary biology↗

bioRxiv2019

Multi-enhancer transcriptional hubs confer phenotypic robustness

We had previously shown in Drosophila melanogaster embryos that low-affinity Ultrabithorax (Ubx)-responsive shavenbaby (svb) enhancers drive robust expression using localized transcriptional environments and that active svb enhancers tended to colocalize, even when placed on different chromosomes (Tsai et al., 2017). Here, we test the hypothesis that these multi-enhancer \"hubs\" improve robustness by increasing transcription factor retention near transcription sites. Deleting a redundant enhancer from the svb locus led to reduced trichome numbers in embryos raised at elevated temperatures. Using high-resolution fluorescence microscopy, we observed lower Ubx concentration and transcriptional output in this deletion allele. Transcription sites of the full svb cis-regulatory region inserted into a different chromosome colocalized with the svb locus, increasing Ubx concentration, the transcriptional output of svb, and partially rescuing the phenotype. Thus, multiple enhancers could reinforce a local transcriptional hub to buffer against environmental stresses and genetic perturbations, providing a mechanism for phenotypical robustness.\n\nImpact statementMultiple enhancers in physical proximity can reinforce shared transcriptional \"hubs\" to retain transcription factors, providing a buffer during environmental stresses and genetic perturbations to preserve phenotypic robustness.

developmental biology↗

bioRxiv2017

Nuclear Microenvironments Modulate Transcription From Low-Affinity Enhancers

Transcription factors regulate gene expression by binding to DNA for short durations and by often binding to low-affinity DNA sequences. It is not clear how such temporally brief, low-affinity interactions can drive efficient transcription. Here we report that the transcription factor Ultrabithorax (Ubx) functionally utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus in nuclear microenvironments of relatively high Ubx concentration. By manipulating the affinity of svb enhancers, we revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. A Ubx cofactor, Homothorax (Hth), was enriched together with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. These results suggest that low affinity sites overcome their kinetic inefficiency by utilizing microenvironments with high concentrations of transcription factors and cofactors. Mechanisms that generate these microenvironments are likely to be a general feature of eukaryotic transcriptional regulation.

cell biology↗

bioRxiv2017

Functional regulatory evolution outside of the minimal even-skipped stripe 2 enhancer

Transcriptional enhancers are regions of DNA that drive gene expression at precise times, levels, and locations. While many studies have elucidated how individual enhancers can evolve, most of this work has focused on what are called \"minimal\" enhancers, the smallest DNA regions that drive expression that approximates an aspect of native gene expression. Here we explore how the Drosophila erecta even-skipped (eve) locus has evolved by testing its activity in the divergent D. melanogaster genome. We found, as has been reported previously, that the minimal D. erecta eve stripe 2 enhancer (eveS2) fails to drive appreciable expression in D. melanogaster [1]. However, we found that a large transgene carrying the entire D. erecta eve locus drives normal eve expression, including in stripe 2. We performed a functional dissection of the region upstream of the D. erecta eveS2 region and found that regulatory information outside of the minimal D. erecta eveS2 contains multiple Zelda motifs that are required for normal expression. Our results illustrate how sequences outside of minimal enhancer regions can evolve functionally through mechanisms other than changes in transcription factor binding sites that drive patterning.

developmental biology↗

bioRxiv2016

Genetic and transgenic reagents for Drosophila simulans, D.mauritiana, D. yakuba, D. santomea and D. virilis

Species of the Drosophila melanogaster species subgroup, including the species D. simulans, D. mauritiana, D. yakuba, and D. santomea, have long served as model systems for studying evolution. Studies in these species have been limited, however, by a paucity of genetic and transgenic reagents. Here we describe a collection of transgenic and genetic strains generated to facilitate genetic studies within and between these species. We have generated many strains of each species containing mapped piggyBac transposons including an enhanced yellow fluorescent protein gene expressed in the eyes and a phiC31 attP site-specific integration site. We have tested a subset of these lines for integration efficiency and reporter gene expression levels. We have also generated a smaller collection of other lines expressing other genetically encoded fluorescent molecules in the eyes and a number of other transgenic reagents that will be useful for functional studies in these species. In addition, we have mapped the insertion locations of 58 transposable elements in D. virilis that will be useful for genetic mapping studies.

genetics↗