A Conserved Developmental Patterning Network Produces Quantitatively Different Output in Multiple Species of Drosophila

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3–4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of equivalent cells

A Statistically Representative Atlas for Mapping Neuronal Circuits in the Drosophila Adult Brain

Published: 23 March 2018The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fninf.2018.00013/full#supplementary-material Supplementary Figure 1. 3D renderings of the 14 regions used for quantitative evaluation of atlas performances in segmentation and registration tasks. The 14 regions shown here were extracted from the atlas of Ito et al. (2014) that has been registered onto the group-wise inter-sex atlas (available from http://fruitfly.tefor.net). Supplementary Figure 2. Selected lines from the Janelia Farm collection showing an overlap value with the search pattern ranking among the first 50 for at least three of the five PDF profiles. (Left) GAL4-driven GFP profile registered on the standard brain. (Right) overlap between the first PDF profile and the GAL4-driven GFP profile. Numbers refer to Janelia Farm lines with associated gene names. Scale bar: 20 μm. Supplementary Table 1. Results of the 3D space query for each of the five PDF profiles. Overlap values are indicated for each Janelia Farm line and the corresponding gene name (FlyBase nomenclature) is indicated for the overlap values ranking among the first 50 for at least three of the five PDF profiles (blue). Bold names correspond to the three lines shown in Figure 10. Supplementary Movie 1. Animated rendering of the group-wise inter-sex atlas. Successively: nc82 template image (2D sections then 3D volume rendering, opaque then transparent); label image (3D surface rendering of anatomical regions, defined following Ito et al. 2014); six registered patterns of GAL4-GFP expression (3D surface rendering of intensity-thresholded pattern images); same patterns (left half of the brain) with the anatomical regions (right half of the brain).Imaging the expression patterns of reporter constructs is a powerful tool to dissect the neuronal circuits of perception and behavior in the adult brain of Drosophila, one of the major models for studying brain functions. To date, several Drosophila brain templates and digital atlases have been built to automatically analyze and compare collections of expression pattern images. However, there has been no systematic comparison of performances between alternative atlasing strategies and registration algorithms. Here, we objectively evaluated the performance of different strategies for building adult Drosophila brain templates and atlases. In addition, we used state-of-the-art registration algorithms to generate a new group-wise inter-sex atlas. Our results highlight the benefit of statistical atlases over individual ones and show that the newly proposed inter-sex atlas outperformed existing solutions for automated registration and annotation of expression patterns. Over 3,000 images from the Janelia Farm FlyLight collection were registered using the proposed strategy. These registered expression patterns can be searched and compared with a new version of the BrainBaseWeb system and BrainGazer software. We illustrate the validity of our methodology and brain atlas with registration-based predictions of expression patterns in a subset of clock neurons. The described registration framework should benefit to brain studies in Drosophila and other insect species.IA-C, TM, NM, FS, and AJ were funded by the Tefor Infrastructure under the Investments for the Future program of the French National Research Agency (Grant #ANR-11-INBS-0014). FR was supported by INSERM. Work at Institut des Neurosciences Paris-Saclay was supported by ANR Infrastructure Tefor and by ANR ClockEye(#ANR-14-CE13-0034-01). JI was supported by the Spanish Ministry of Economy and Competitiveness (TEC2014-51882-P), the European Union's Horizon 2020 research and innovation programme (Marie Sklodowska-Curie grant 654911, project THALAMODEL), and the European Research Council (ERC Starting Grant no. 677697 BUNGEE-TOOLS). VRVis (KB, FS) is funded by BMVIT, BMWFW, Styria, SFG and Vienna Business Agency in the scope of COMET - Competence Centers for Excellent Technologies (854174) which is managed by FFG. The Institut Jean-Pierre Bourgin benefits from the support of the LabEx Saclay Plant Sciences-SPS (#ANR-10-LABX-0040-SPS)

Expression of Distal-less, dachshund, and optomotor blind in Neanthes arenaceodentata (Annelida, Nereididae) does not support homology of appendage-forming mechanisms across the Bilateria

The similarity in the genetic regulation of arthropod and vertebrate appendage formation has been interpreted as the product of a plesiomorphic gene network that was primitively involved in bilaterian appendage development and co-opted to build appendages (in modern phyla) that are not historically related as structures. Data from lophotrochozoans are needed to clarify the pervasiveness of plesiomorphic appendage forming mechanisms. We assayed the expression of three arthropod and vertebrate limb gene orthologs, Distal-less (Dll), dachshund (dac), and optomotor blind (omb), in direct-developing juveniles of the polychaete Neanthes arenaceodentata. Parapodial Dll expression marks premorphogenetic notopodia and neuropodia, becoming restricted to the bases of notopodial cirri and to ventral portions of neuropodia. In outgrowing cephalic appendages, Dll activity is primarily restricted to proximal domains. Dll expression is also prominent in the brain. dac expression occurs in the brain, nerve cord ganglia, a pair of pharyngeal ganglia, presumed interneurons linking a pair of segmental nerves, and in newly differentiating mesoderm. Domains of omb expression include the brain, nerve cord ganglia, one pair of anterior cirri, presumed precursors of dorsal musculature, and the same pharyngeal ganglia and presumed interneurons that express dac. Contrary to their roles in outgrowing arthropod and vertebrate appendages, Dll, dac, and omb lack comparable expression in Neanthes appendages, implying independent evolution of annelid appendage development. We infer that parapodia and arthropodia are not structurally or mechanistically homologous (but their primordia might be), that Dll’s ancestral bilaterian function was in sensory and central nervous system differentiation, and that locomotory appendages possibly evolved from sensory outgrowths

Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution I: data acquisition pipeline

BACKGROUND: To model and thoroughly understand animal transcription networks, it is essential to derive accurate spatial and temporal descriptions of developing gene expression patterns with cellular resolution. RESULTS: Here we describe a suite of methods that provide the first quantitative three-dimensional description of gene expression and morphology at cellular resolution in whole embryos. A database containing information derived from 1,282 embryos is released that describes the mRNA expression of 22 genes at multiple time points in the Drosophila blastoderm. We demonstrate that our methods are sufficiently accurate to detect previously undescribed features of morphology and gene expression. The cellular blastoderm is shown to have an intricate morphology of nuclear density patterns and apical/basal displacements that correlate with later well-known morphological features. Pair rule gene expression stripes, generally considered to specify patterning only along the anterior/posterior body axis, are shown to have complex changes in stripe location, stripe curvature, and expression level along the dorsal/ventral axis. Pair rule genes are also found to not always maintain the same register to each other. CONCLUSION: The application of these quantitative methods to other developmental systems will likely reveal many other previously unknown features and provide a more rigorous understanding of developmental regulatory networks

Functional Evolution of a cis-Regulatory Module

Lack of knowledge about how regulatory regions evolve in relation to their structure–function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module—the even-skipped stripe 2 enhancer—from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure–function, mechanisms of speciation and computational identification of regulatory modules

Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution I: data acquisition pipeline

BACKGROUND: To model and thoroughly understand animal transcription networks, it is essential to derive accurate spatial and temporal descriptions of developing gene expression patterns with cellular resolution. RESULTS: Here we describe a suite of methods that provide the first quantitative three-dimensional description of gene expression and morphology at cellular resolution in whole embryos. A database containing information derived from 1,282 embryos is released that describes the mRNA expression of 22 genes at multiple time points in the Drosophila blastoderm. We demonstrate that our methods are sufficiently accurate to detect previously undescribed features of morphology and gene expression. The cellular blastoderm is shown to have an intricate morphology of nuclear density patterns and apical/basal displacements that correlate with later well-known morphological features. Pair rule gene expression stripes, generally considered to specify patterning only along the anterior/posterior body axis, are shown to have complex changes in stripe location, stripe curvature, and expression level along the dorsal/ventral axis. Pair rule genes are also found to not always maintain the same register to each other. CONCLUSION: The application of these quantitative methods to other developmental systems will likely reveal many other previously unknown features and provide a more rigorous understanding of developmental regulatory networks

mRNA diffusion explains protein gradients in Drosophila early development

We propose a new model describing the production and the establishment of the stable gradient of the Bicoid protein along the antero-posterior axis of the embryo of _Drosophila_. In this model, we consider that _bicoid_ mRNA diffuses along the antero-posterior axis of the embryo and the protein is produced in the ribosomes localized near the syncytial nuclei. Bicoid protein stays localized near the syncytial nuclei as observed in experiments.We calibrate the parameters of the mathematical model with experimental data taken during the cleavage stages 11 to 14 of the developing embryo of _Drosophila_. We obtain good agreement between the experimental and the model gradients, with relative errors in the range 5-8%. The inferred diffusion coefficient of _bicoid_ mRNA is in the range 4.6 x 10^-12^ - 1.5 x10^-11^ m^2^s^-1^, in agreement with the theoretical predictions and experimental measurements for the diffusion of macromolecules in the cytoplasm. We show that the model based on the mRNA diffusion hypothesis is consistent with the known observational data, supporting the recent experimental findings of the gradient of _bicoid_ mRNA in _Drosophila_ [Spirov _et al._ (2009) _Development_ 136:605-614]

FlyEx, the quantitative atlas on segmentation gene expression at cellular resolution

The datasets on gene expression are the valuable source of information about the functional state of an organism. Recently, we have acquired the large dataset on expression of segmentation genes in the Drosophila blastoderm. To provide efficient access to the data, we have developed the FlyEx database (http://urchin.spbcas.ru/flyex). FlyEx contains 4716 images of 14 segmentation gene expression patterns obtained from 1579 embryos and 9 500 000 quantitative data records. Reference data are available for all segmentation genes in cycles 11–13 and all temporal classes of cycle 14A. FlyEx supports operations on images of gene expression patterns. The database can be used to examine the quality of data, analyze the dynamics of formation of segmentation gene expression domains, as well as to estimate the variability of gene expression patterns. Currently, a user is able to monitor and analyze the dynamics of formation of segmentation gene expression domains over the whole period of segment determination, that amounts to 1.5 h of development. FlyEx supports the data downloads and construction of personal reference datasets, that makes it possible to more effectively use and analyze data