@article {69385, title = {A Bayesian Solution to Count the Number of Molecules within a Diffraction Limited Spot}, journal = {bioRxiv}, year = {2024}, month = {2024 Apr 22}, abstract = {

We address the problem of inferring the number of independently blinking fluorescent light emitters, when only their combined intensity contributions can be observed at each timepoint. This problem occurs regularly in light microscopy of objects that are smaller than the diffraction limit, where one wishes to count the number of fluorescently labelled subunits. Our proposed solution directly models the photo-physics of the system, as well as the blinking kinetics of the fluorescent emitters as a fully differentiable hidden Markov model. Given a trace of intensity over time, our model jointly estimates the parameters of the intensity distribution per emitter, their blinking rates, as well as a posterior distribution of the total number of fluorescent emitters. We show that our model is consistently more accurate and increases the range of countable subunits by a factor of two compared to current state-of-the-art methods, which count based on autocorrelation and blinking frequency, Further-more, we demonstrate that our model can be used to investigate the effect of blinking kinetics on counting ability, and therefore can inform experimental conditions that will maximize counting accuracy.

}, doi = {10.1101/2024.04.18.590066}, url = {https://www.biorxiv.org/content/early/2024/04/22/2024.04.18.590066}, author = {Alexander Hillsley and Johannes Stein and Paul W. Tillberg and David L. Stern and Jan Funke} } @article {68919, title = {The density of regulatory information is a major determinant of evolutionary constraint on non-coding DNA in Drosophila}, journal = {Molecular Biology and Evolution}, volume = {41}, year = {2024}, month = {2024 Feb 01}, pages = {msae004}, abstract = {

The density and distribution of regulatory information in non-coding DNA of eukaryotic genomes is largely unknown. Evolutionary analyses have estimated that \~{}60\% of nucleotides in intergenic regions of the\ D. melanogaster\ genome is functionally relevant. This estimate is difficult to reconcile with the commonly accepted idea that enhancers are compact regulatory elements that generally encompass less than 1 kilobase of DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene\ shavenbaby\ (svb). Most of the \~{}90 kilobases of this large regulatory region is highly conserved in the genus\ Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of\ svb\ in different contexts of\ Drosophila\ development, we found that the regulatory architecture that drives\ svb\ expression in the abdominal pupal epidermis is organized in a dramatically different way than the information that drives\ svb\ expression in the embryonic epidermis. While in the embryonic epidermis\ svb\ is activated by compact and dispersed enhancers,\ svb\ expression in the pupal epidermis is driven by large regions with enhancer activity, which occupy a great portion of the\ svb cis-regulatory DNA. We observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved non-coding DNA of the\ Drosophila\ genome is contained within putative regulatory DNA. These results suggest that part of the evolutionary constraint on non-coding DNA of\ Drosophila\ is explained by the density of regulatory information.

}, doi = {10.1093/molbev/msae004}, url = {https://doi.org/10.1093/molbev/msae004}, author = {Gonzalo Sabar{\'\i}s and Daniela M. Ort{\'\i}z and Ian Laiker and Ignacio Mayansky and Sujay Naik and Giacomo Cavalli and David L. Stern and Ella Preger-Ben Noon and Nicol{\'a}s Frankel} } @article {69008, title = {Nested neural circuits generate distinct acoustic signals during Drosophila courtship}, journal = {Current Biology}, volume = {34}, year = {2024}, month = {2024 Feb 26}, pages = {808-24}, abstract = {

Many motor control systems generate multiple movements using a common set of muscles. How are premotor circuits able to flexibly generate diverse movement patterns? Here, we characterize the neuronal circuits that drive the distinct courtship songs of\ Drosophila melanogaster. Male flies vibrate their wings towards females to produce two different song modes {\textendash} pulse and sine song {\textendash} which signal species identity and male quality. Using cell-type specific genetic reagents and the connectome, we provide a cellular and synaptic map of the circuits in the male ventral nerve cord that generate these songs and examine how activating or inhibiting each cell type within these circuits affects the song. Our data reveal that the song circuit is organized into two nested feed-forward pathways, with extensive reciprocal and feed-back connections. The larger network produces pulse song, the more complex and ancestral song form. A subset of this network produces sine song, the simpler and more recent form. Such nested organization may be a common feature of motor control circuits in which evolution has layered increasing flexibility on to a basic movement pattern.

}, doi = {10.1016/j.cub.2024.01.015}, url = {https://doi.org/10.1016/j.cub.2024.01.015}, author = {Joshua L. Lillvis and Kaiyu Wang and Hiroshi M. Shiozaki and Min Xu and David L. Stern and Barry J. Dickson} } @article {69010, title = {Olfactory sensory neuron population expansions influence projection neuron adaptation and enhance odour tracking}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 Aug 15}, pages = {7041}, abstract = {

The evolutionary expansion of sensory neuron populations detecting important environmental cues is widespread, but functionally enigmatic. We investigated this phenomenon through comparison of homologous olfactory pathways of Drosophila melanogaster and its close relative Drosophila sechellia, an extreme specialist for Morinda citrifolia noni fruit. D. sechellia has evolved species-specific expansions in select, noni-detecting olfactory sensory neuron (OSN) populations, through multigenic changes. Activation and inhibition of defined proportions of neurons demonstrate that OSN number increases contribute to stronger, more persistent, noni-odour tracking behaviour. These expansions result in increased synaptic connections of sensory neurons with their projection neuron (PN) partners, which are conserved in number between species. Surprisingly, having more OSNs does not lead to greater odour-evoked PN sensitivity or reliability. Rather, pathways with increased sensory pooling exhibit reduced PN adaptation, likely through weakened lateral inhibition. Our work reveals an unexpected functional impact of sensory neuron population expansions to explain ecologically-relevant, species-specific behaviour.

}, doi = {10.1038/s41467-024-50808-w}, url = {https://doi.org/10.1038/s41467-024-50808-w}, author = {Suguru Takagi and Liliane Abuin and S. David Stupski and J. Roman Arguello and Lucia Prieto-Godino and David L. Stern and Steeve Cruchet and Raquel {\'A}lvarez-Oca{\~n}a and Carl F. R. Wienecke and Floris van Breugel and Thomas O. Auer and Richard Benton} } @article {69253, title = {Sensory neuron population expansion enhances odor tracking without sensitizing projection neurons}, journal = {bioRxiv}, year = {2024}, month = {2024 Feb 28}, abstract = {

The evolutionary expansion of sensory neuron populations detecting important environmental cues is widespread, but functionally enigmatic. We investigated this phenomenon through comparison of homologous neural pathways of Drosophila melanogaster and its close relative Drosophila sechellia, an extreme specialist for Morinda citrifolia noni fruit. D. sechellia has evolved species-specific expansions in select, noni-detecting olfactory sensory neuron (OSN) populations, through multigenic changes. Activation and inhibition of defined proportions of neurons demonstrate that OSN population increases contribute to stronger, more persistent, noni-odor tracking behavior. These sensory neuron expansions result in increased synaptic connections with their projection neuron (PN) partners, which are conserved in number between species. Surprisingly, having more OSNs does not lead to greater odor-evoked PN sensitivity or reliability. Rather, pathways with increased sensory pooling exhibit reduced PN adaptation, likely through weakened lateral inhibition. Our work reveals an unexpected functional impact of sensory neuron expansions to explain ecologically-relevant, species-specific behavior.

}, doi = {10.1101/2023.09.15.556782}, url = {https://www.biorxiv.org/content/early/2024/02/28/2023.09.15.556782}, author = {Suguru Takagi and Gizem Sancer and Liliane Abuin and S. David Stupski and J. Roman Arguello and Lucia L. Prieto-Godino and David L. Stern and Steeve Cruchet and Raquel {\'A}lvarez-Oca{\~n}a and Carl F. R. Wienecke and Floris van Breugel and James M. Jeanne and Thomas O. Auer and Richard Benton} } @article {69146, title = {Song Torrent: A modular, open-source 96-chamber audio and video recording apparatus with optogenetic activation and inactivation capabilities for Drosophila}, journal = {bioRxiv}, year = {2024}, month = {2024 Jan 10}, abstract = {

Background

Results

Implications

}, doi = {10.1101/2024.01.09.574712}, url = {https://doi.org/10.1101/2024.01.09.574712}, author = {Steve Sawtelle and Lakshmi Narayan and Yun Ding and Elizabeth Kim and Emily L. Behrman and Joshua L. Lillvis and Takashi Kawase and David L. Stern} } @article {68974, title = {Combinatorial circuit dynamics orchestrate flexible motor patterns in Drosophila.}, journal = {bioRxiv}, year = {2023}, month = {2023 Sep 07}, abstract = {

Motor systems flexibly implement diverse motor programs to pattern behavioral sequences, yet their neural underpinnings remain unclear. Here, we investigated the neural circuit mechanisms of flexible courtship behavior in\ Drosophila. Courting males alternately produce two types of courtship song. By recording calcium signals in the ventral nerve cord (VNC) in behaving flies, we found that different songs are produced by activating overlapping neural populations with distinct motor functions in a combinatorial manner. Recordings from the brain suggest that song is driven by two descending pathways {\textendash} one defines when to sing and the other specifies what song to sing. Connectomic analysis reveals that these {\textquotedblleft}when{\textquotedblright} and {\textquotedblleft}what{\textquotedblright} descending pathways provide structured input to VNC neurons with different motor functions. These results suggest that dynamic changes in the activation patterns of descending pathways drive different combinations of motor modules, thereby flexibly switching between different motor actions.

}, doi = {10.1101/2022.12.14.520499}, url = {https://doi.org/10.1101/2022.12.14.520499}, author = {Hiroshi M. Shiozaki and Kaiyu Wang and Joshua L. Lillvis and Min Xu and Barry J. Dickson and David L. Stern} } @article {68832, title = {The Janelia Atalanta plasmids provide a simple and efficient CRISPR/Cas9-mediated homology directed repair platform for Drosophila}, journal = {bioRxiv}, year = {2023}, month = {2023 Jun 17}, abstract = {

Homology-directed repair (HDR) is a powerful tool for modifying genomes in precise ways to address many biological questions. Use of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-Cas9 induced targeted DNA double-strand breakage has substantially simplified use of homology-directed repair to introduce specific perturbations in\ Drosophila, but existing platforms for CRISPR-Cas9-mediated HDR in\ Drosophila\ involve multiple cloning steps and have low efficiency. To simplify cloning of HDR plasmids, we designed a new plasmid platform, the Janelia Atalanta (pJAT) series, that exploits recent advances in dsDNA synthesis to facilitate Gateway cloning of gRNA sequences and homology arms in one step. Surprisingly, the pJAT plasmids yielded considerably higher HDR efficiency (approximately 25\%) than we have observed with other approaches. pJAT plasmids work in multiple\ Drosophila\ species and exhibited such high efficiency that previously impossible experiments in\ Drosophila, such as driving targeted chromosomal inversions, were made possible. We provide pJAT plasmids for a range of commonly performed experiments including targeted insertional mutagenesis, insertion of phiC31-mediated attP landing sites, generation of strains carrying a germ-line source of Cas9, and induction of chromosomal rearrangements. We also provide {\textquotedblleft}empty{\textquotedblright} pJAT plasmids with multiple cloning sites to simplify construction of plasmids with new functionality. The pJAT platform is generic and may facilitate improved efficiency CRISPR-Cas9 HDR in a wide range of model and non-model organisms.

}, doi = {10.1101/2023.06.17.545412}, url = {https://doi.org/10.1101/2023.06.17.545412}, author = {David L. Stern and Elizabeth Kim and Emily L. Behrman} } @article {68966, title = {The Neural Basis of Drosophila Courtship Song}, journal = {bioRxiv}, year = {2023}, month = {2023 Sep 01}, abstract = {

Animal sounds are produced by patterned vibrations of specific organs, but the neural circuits that drive these vibrations are not well defined in any animal. Here we provide a functional and synaptic map of most of the neurons in the\ Drosophila\ male ventral nerve cord (the analog of the vertebrate spinal cord) that drive complex, patterned song during courtship. Male\ Drosophila\ vibrate their wings toward females during courtship to produce two distinct song modes {\textendash} pulse and sine song {\textendash} with characteristic features that signal species identity and male quality. We identified song-producing neural circuits by optogenetically activating and inhibiting identified cell types in the ventral nerve cord (VNC) and by tracing their patterns of synaptic connectivity in the male VNC connectome. The core song circuit consists of at least eight cell types organized into overlapping circuits, where all neurons are required for pulse song and a subset are required for sine song. The pulse and sine circuits each include a feed-forward pathway from brain descending neurons to wing motor neurons, with extensive reciprocal and feed-back connections. We also identify specific neurons that shape the individual features of each song mode. These results reveal commonalities amongst diverse animals in the neural mechanisms that generate diverse motor patterns from a single set of muscles.

}, doi = {10.1101/2023.08.30.555537}, url = {https://doi.org/10.1101/2023.08.30.555537}, author = {Joshua L. Lillvis and Kaiyu Wang and Hiroshi M. Shiozaki and Min Xu and David L. Stern and Barry J. Dickson} } @article {68117, title = {An essential experimental control for functional connectivity mapping with optogenetics.}, journal = {bioRxiv}, year = {2022}, month = {2022 May 28}, abstract = {

To establish functional connectivity between two candidate neurons that might form a circuit element, a common approach is to activate an optogenetic tool such as Chrimson in the candidate pre-synaptic neuron and monitor fluorescence of the calcium-sensitive indicator GCaMP in a candidate post-synaptic neuron. While performing such experiments, we found that low levels of leaky Chrimson expression can lead to strong artifactual GCaMP signals in presumptive postsynaptic neurons even when Chrimson is not intentionally expressed in any particular neurons. Withholding all-trans\ retinal, the chromophore required as a co-factor for Chrimson response to light, eliminates GCaMP signal but does not provide an experimental control for leaky Chrimson expression. Leaky Chrimson expression appears to be an inherent feature of current Chrimson transgenes, since artifactual connectivity was detected with Chrimson transgenes integrated into three different genomic locations (two insertions tested in larvae; a third insertion tested in the adult fly). These false-positive signals may complicate the interpretation of functional connectivity experiments. We illustrate how a no-Gal4 negative control improves interpretability of functional connectivity assays. We also propose a simple but effective procedure to identify experimental conditions that minimize potentially incorrect interpretations caused by leaky Chrimson expression.

}, doi = {10.1101/2022.05.26.493610}, url = {https://doi.org/10.1101/2022.05.26.493610}, author = {David Tadres and Hiroshi M. Shiozaki and Ibrahim Tastekin and David L. Stern and Matthieu Louis} } @article {68569, title = {Neural coding of distinct motor patterns during Drosophila courtship song}, journal = {bioRxiv}, year = {2022}, month = {2022 Dec 15}, abstract = {

Animals flexibly switch between different actions by changing neural activity patterns for motor control. Courting\ Drosophila melanogaster\ males produce two different acoustic signals, pulse and sine song, each of which can be promoted by artificial activation of distinct neurons. However, how the activity of these neurons implements flexible song production is unknown. Here, we developed an assay to record neuronal calcium signals in the ventral nerve cord, which contains the song motor circuit, in singing flies. We found that sine-promoting neurons, but not pulse-promoting neurons, show strong activation during sine song. In contrast, both pulse- and sine-promoting neurons are active during pulse song. Furthermore, population calcium imaging in the song circuit suggests that sine song involves activation of a subset of neurons that are also active during pulse song. Thus, differential activation of overlapping, rather than distinct, neural populations underlies flexible motor actions during acoustic communication in\ D. melanogaster.

}, doi = {10.1101/2022.12.14.520499}, url = {https://doi.org/10.1101/2022.12.14.520499}, author = {Hiroshi M. Shiozaki and Kaiyu Wang and Joshua L. Lillvis and Min Xu and Barry J. Dickson and David L. Stern} } @article {45800, title = {Molecular characterization of pea aphid facultative parthenogenesis}, journal = {Developmental Biology}, volume = {319}, year = {2008}, month = {07/2008}, pages = {494-495}, abstract = {

Meiosis is a highly conserved process in which a diploid genome is recombined and assorted into haploid gametes. Remarkably, the pea aphid Acyrthosiphon pisum exhibits a reproductive polyphenism whereby environmental signals trigger a switch between apomixis (parthenogenetic reproduction) and meiosis (sexual reproduction). Aphid apomixis results in daughter embryo clones with 2n genome content without male contribution or recombination. This important adaptation allows aphid populations to not only rapidly expand upon abundant resources during summer but also survive winter. How aphids have evolved this ability to switch between parthenogenesis and sexual meiosis is unknown. To arrive at a mechanistic explanation for this developmental plasticity, I determined meiosis gene activity in sexuals and asexuals. I first identified homologs of a core set of meiosis genes from the pea aphid genome. Next, I tested the expression of these core meiosis genes by PCR spanning across at least one intron from cDNA isolated from asexual and sexual ovaries. Surprisingly, meiosis specific genes (e.g., Spo11, Msh4, Msh5, Hop2 and Mnd1) are expressed in asexual ovaries. Additionally, the Spo11 PCR product contained intronic sequence, thus representing unspliced mRNA. Future experiments looking at the quantities and localizations of mRNA and protein will help to distinguish among several possible explanations for these results. Further molecular characterization of this phenotypic plasticity will be helpful in understanding how multiple interacting pathways can evolve to create alternate developmental phenotypes.

}, doi = {10.1016/j.ydbio.2008.05.098}, author = {Dayalan G. Srinivasan and Greg K. Davis and David L. Stern} } @article {45807, title = {The evolution of sociality in aphids: a clone{\textquoteright}s-eye view}, journal = {The evolution of social behavior in insects and arachnids.}, year = {1997}, month = {03/1997}, pages = {150-165}, abstract = {

A number of aphid species produce individuals, termed soldiers, that defend the colony by attacking predators. Soldiers have either reduced or zero direct reproductive fitness. Their behavior is therefore altruistic in the classical sense: an individual is behaving in a way that incurs reproductive costs on itself and confers reproductive benefits on another. However, comparison with the better{\textendash}known eusocial insects (Hymenoptera, Isoptera) indicates that there are important differences between clonal and sexual social animals.

Here we take a clone{\textquoteright}s{\textendash}eye view and conclude that many facets of aphid sociality are best thought of in terms of resource allocation: for example, the choice between investment in defense and reproduction. This view considerably simplifies some aspects of the problem and highlights the qualitatively different nature of genetic heterogeneity in colonies of aphids and of other social insects. In sexually reproducing social insects, each individual usually has a different genome, which leads to genetic conflicts of interest between individuals. In social aphids, all members of a clone have identical genomes, barring new mutations, and there should be no disagreement among clonemates about investment decisions. Genetic heterogeneity within colonies can arise, but principally through clonal mixing, and this means that investment decisions will vary between different clones rather than among all individuals.

}, isbn = {9780511721953}, doi = {10.1017/CBO9780511721953.008}, url = {http://ebooks.cambridge.org/chapter.jsf?bid=CBO9780511721953\&cid=CBO9780511721953A015}, author = {David L. Stern and William A. Foster} } @article {45814, title = {A phylogenetic reanalysis of allozyme variation among populations of Galapagos finches}, journal = {Zoological Journal of the Linnean Society}, volume = {118}, year = {1996}, pages = {119-134}, abstract = {

We reanalysed Yang \& Pattern{\textquoteright}s allozyme data, published in Auk in 1981, of Darwin{\textquoteright}s finches with a variety of distance and cladistic methods to estimate the phylogeny of the group. Different methods yielded different results, nevertheless there was widespread agreement among the distance methods on several groupings. First, the two species of Camarhynchus grouped near one another, but not always as a monophyletic group. Second, Cactospiza pallida and Platyspiza crassirostris formed a monophyletic group. Finally, all the methods (including parsimony) supported the monophyly of the ground finches. The three distance methods also found close relationships generally between each of two populations of Geospiza scandens, G. difficilis and G. conirostris. There is evidence for inconstancy of evolutionary rates among species. Results from distance methods allowing for rate variation among lineages suggest three conclusions which differ from Yang and Patton{\textquoteright}s findings. First, the monophyletic ground finches arose from the paraphyletic tree finches. Yang and Patton found that the ground finches and tree finches were sister monophyletic taxa. Second, Geospiza scandens appears to be a recently derived species, and not the most basal ground finch. Third, G. fuliginosa is not a recently derived species of ground finch, but was derived from an older split from the remaining ground finches. Most of these conclusions should be considered tentative both because the parsimony trees disagreed sharply with the distance trees and because no clades were strongly supported by the results of bootstrapping and statistical tests of alternative hypotheses. Absence of strong support for clades was probably due to insufficient data. Future phylogenetic studies, preferably using DNA sequence data from several unlinked loci, should sample several populations of each species, and should attempt to assess the importance of hybridization in species phylogeny.

}, doi = {10.1111/j.1096-3642.1996.tb00222.x}, author = {David L. Stern and Peter R. Grant} }