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Detecting mosaic patterns in phenotypic disparity

Evolutionary biologists have long sought to understand the full complexity in pattern and process that shapes organismal diversity. Although phylogenetic comparative methods are often used to reconstruct complex evolutionary dynamics, they are typically limited to a single phenotypic trait. Extensions that accommodate multiple traits lack the ability to partition multidimensional datasets into a set of mosaic suites of evolutionarily linked characters. I introduce a comparative framework that identifies heterogeneity in evolutionary patterns across large datasets of continuous traits. Using a model of continuous trait evolution based on the differential accumulation of disparity across lineages in a phylogeny, the approach algorithmically partitions traits into a set of character suites that best explains the data, where each suite displays a distinct pattern in phylogenetic morphological disparity. When applied to empirical data, the approach revealed a mosaic pattern predicted by developmental biology. The evolutionary distinctiveness of individual suites can be investigated in more detail, either by fitting conventional comparative models or by directly studying the phylogenetic patterns in disparity recovered during the analysis. This framework can supplement existing comparative approaches by inferring the complex, integrated patterns that shape evolution across the body plan from disparate developmental, morphometric, and environmental sources of phenotypic data.

evolutionary biology

Tissue-specific tagging of endogenous loci in Drosophila melanogaster

Fluorescent protein tags have revolutionized cell and developmental biology, and in combination with binary expression systems they enable diverse tissue-specific studies of protein function. However these binary expression systems often do not recapitulate endogenous protein expression levels, localization, binding partners, and developmental windows of gene expression. To address these limitations, we have developed a method called T-STEP (Tissue-Specific Tagging of Endogenous Proteins) that allows endogenous loci to be tagged in a tissue specific manner. T-STEP uses a combination of efficient gene targeting and tissue-specific recombinase-mediated tag swapping to temporally and spatially label endogenous proteins. We have employed this method to GFP tag OCRL (a phosphoinositide-5-phosphatase in the endocytic pathway) and Vps35 (a Parkinson's disease-implicated component of the endosomal retromer complex) in diverse Drosophila tissues including neurons, glia, muscles, and hemocytes. Selective tagging of endogenous proteins allows for the first time cell type-specific live imaging and proteomics in complex tissues.

Neuroscience

Massively parallel single cell lineage tracing using CRISPR/Cas9 induced genetic scars

A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many different cell types. Single-cell RNA-sequencing (scRNA-seq) has become a widely-used method due to its ability to identify all cell types in a tissue or organ in a systematic manner 1-3. However, a major challenge is to organize the resulting taxonomy of cell types into lineage trees revealing the developmental origin of cells. Here, we present a strategy for simultaneous lineage tracing and transcriptome profiling in thousands of single cells. By combining scRNA-seq with computational analysis of lineage barcodes generated by genome editing of transgenic reporter genes, we reconstruct developmental lineage trees in zebrafish larvae and adult fish. In future analyses, LINNAEUS (LINeage tracing by Nuclease-Activated Editing of Ubiquitous Sequences) can be used as a systematic approach for identifying the lineage origin of novel cell types, or of known cell types under different conditions.

systems biology

What to compare and how: comparative transcriptomics for Evo-Devo

Evolutionary developmental biology has grown historically from the capacity to relate patterns of evolution in anatomy to patterns of evolution of expression of specific genes, whether between very distantly related species, or very closely related species or populations. Scaling up such studies by taking advantage of modern transcriptomics brings promising improvements, allowing us to estimate the overall impact and molecular mechanisms of convergence, constraint or innovation in anatomy and development. But it also presents major challenges, including the computational definitions of anatomical homology and of organ function, the criteria for the comparison of developmental stages, the annotation of transcriptomics data to proper anatomical and developmental terms, and the statistical methods to compare transcriptomic data between species to highlight significant conservation or changes. In this article, we review these challenges, and the ongoing efforts to address them, which are emerging from bioinformatics work on ontologies, evolutionary statistics, and data curation, with a focus on their implementation in the context of the development of our database Bgee (http://bgee.org).

Evolutionary Biology

How domain growth is implemented determines the long term behaviour of a cell population through its effect on spatial correlations.

Domain growth plays an important role in many biological systems, and so the inclusion of domain growth in models of these biological systems is important to understanding how these biological systems function. In this work we present methods to include the effects of domain growth on the evolution of spatial correlations in a continuum approximation of a lattice-based model of cell motility and proliferation. We show that, depending on the way in which domain growth is implemented, different steady-state densities are predicted for an agent population. Furthermore, we demonstrate that the way in which domain growth is implemented can result in the evolution of the agent density depending on the size of the domain. Continuum approximations that ignore spatial correlations cannot capture these behaviours, while those that account for spatial correlations do. These results will be of interest to researchers in developmental biology, as they suggest that the nature of domain growth can determine the characteristics of cell populations.

Systems Biology

Manuscript 101: A Data-Driven Writing Exercise For Beginning Scientists

Learning to write a scientific manuscript is one of the most important and rewarding scientific training experiences, yet most young scientists only embark on this experience relatively late in graduate school, after gathering sufficient data in the lab. Yet, familiarity with the process of writing a scientific manuscript and receiving peer reviews, often leads to a more focused and driven experimental approach. To jump-start this training, we developed a protocol for teaching manuscript writing and reviewing in the classroom, appropriate for new graduate or upper-level undergraduate students of developmental biology. First, students are provided one of four cartoon data sets, which are focused on genetic models of animal development. Students are instructed to use their creativity to convert evidence into argument, and then to integrate their interpretations into a manuscript, including an illustrated, mechanistic model figure. After student manuscripts are submitted, manuscripts are redacted and distributed to classmates for peer review. Here, we present our cartoon datasets, homework instructions, and grading rubrics as a new resource for the scientific community. We also describe methods for developing new datasets so that instructors can adapt this activity to other disciplines. Our data-driven manuscript writing exercise, as well as the formative and summative assessments resulting from the peer review, enables students to learn fundamental concepts in developmental genetics. In addition, students practice essential skills of scientific communication, including arguing from evidence, developing and testing models, the unique conventions of scientific writing, and the joys of scientific story telling.

scientific communication and education

Pollen Patterns Form from Modulated Phases

Pollen grains are known for their impressive variety of species-specific, microscale surface patterning. Despite having similar biological developmental steps, pollen grain surface features are remarkably geometrically varied. Previous work suggests that a physical process may drive this pattern formation and that the observed diversity of patterns can be explained by viewing pollen pattern development as a phase transition to a spatially modulated phase. Several studies have shown that the polysaccharide material of plant cell walls undergoes phase separation in the absence of cross-linking stabilizers of the mixed phase. Here we show experimental evidence that phase separation of the extracellular polysaccharide material (primexine) during pollen cell development leads to a spatially modulated phase. The spatial pattern of this phase-separated primexine is also mechanically coupled to the undulation of the pollen cell membrane. The resulting patterned pools of denser primexine form the negative template of the ultimate sites of sporopollenin deposition, leading to the final micropattern observed in the mature pollen. We then present a general physical model of pattern formation via modulated phases. Using analytical and numerical techniques, we find that most of the pollen micropatterns observed in biological evolution could result from a physical process of modulated phases. However, an analysis of the relative rates of transitions from states that are equilibrated to or from states that are not equilibrated suggests that while equilibrium states of this process have occurred throughout evolutionary history, there has been no particular evolutionary selection for symmetric, equilibrated states.

biophysics

Hemodynamic forces can be accurately measured in vivo with optical tweezers

Force sensing and generation at the tissular and cellular scale is central to many biological events. There is a growing interest in modern cell biology for methods enabling force measurements in vivo. Optical trapping allows non-invasive probing of pico-Newton forces and thus emerged as a promising mean for assessing biomechanics in vivo. Nevertheless, the main obstacles rely in the accurate determination of the trap stiffness in heterogeneous living organisms, at any position where the trap is used. A proper calibration of the trap stiffness is thus required for performing accurate and reliable force measurements in vivo. Here, we introduce a method that overcomes these difficulties by accurately measuring hemodynamic profiles in order to calibrate the trap stiffness. Doing so, and using numerical methods to assess the accuracy of the experimental data, we measured flow profiles and drag forces imposed to trapped red blood cells of living zebrafish embryos. Using treatments enabling blood flow tuning, we demonstrated that such method is powerful in measuring hemodynamic forces in vivo with high accuracy and confidence. Altogether, this study demonstrates the power of optical tweezing in measuring low range hemodynamic forces in vivo and offers an unprecedented tool in both cell and developmental biology.

biophysics

Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing

Conventional methods for profiling the molecular content of biological samples fail to resolve heterogeneity that is present at the level of single cells. In the past few years, single cell RNA sequencing has emerged as a powerful strategy for overcoming this challenge. However, its adoption has been limited by a paucity of methods that are at once simple to implement and cost effective to scale massively. Here, we describe a combinatorial indexing strategy to profile the transcriptomes of large numbers of single cells or single nuclei without requiring the physical isolation of each cell (Single cell Combinatorial Indexing RNA-seq or sci-RNA-seq). We show that sci-RNA-seq can be used to efficiently profile the transcriptomes of tens-of-thousands of single cells per experiment, and demonstrate that we can stratify cell types from these data. Key advantages of sci-RNA-seq over contemporary alternatives such as droplet-based single cell RNA-seq include sublinear cost scaling, a reliance on widely available reagents and equipment, the ability to concurrently process many samples within a single workflow, compatibility with methanol fixation of cells, cell capture based on DNA content rather than cell size, and the flexibility to profile either cells or nuclei. As a demonstration of sci-RNA-seq, we profile the transcriptomes of 42,035 single cells from C. elegans at the L2 stage, effectively 50-fold \"shotgun cellular coverage\" of the somatic cell composition of this organism at this stage. We identify 27 distinct cell types, including rare cell types such as the two distal tip cells of the developing gonad, estimate consensus expression profiles and define cell-type specific and selective genes. Given that C. elegans is the only organism with a fully mapped cellular lineage, these data represent a rich resource for future methods aimed at defining cell types and states. They will advance our understanding of developmental biology, and constitute a major step towards a comprehensive, single-cell molecular atlas of a whole animal.

genomics

Deep, Staged Transcriptomic Resources for the Novel Coleopteran Models Atrachya menetriesi and Callosobruchus maculatus

Despite recent efforts to sample broadly across metazoan and insect diversity, current sequence resources in the Coleoptera do not adequately describe the diversity of the clade. Here we present deep, staged transcriptomic data for two coleopteran species, Atrachya menetriesi (Faldermann 1835) and Callosobruchus maculatus (Fabricius 1775). Our sampling covered key stages in ovary and early embryonic development in each species. We utilized this data to build combined assemblies for each species which were then analysed in detail. The combined A. menetriesi assembly consists of 228,096 contigs with an N50 of 1,598 bp, while the combined C. maculatus assembly consists of 128,837 contigs with an N50 of 2,263 bp. For these assemblies, 34.6% and 32.4% of contigs were identified using Blast2GO, and 97% and 98.3% of the BUSCO set of metazoan orthologs were present, respectively. We also carried out manual annotation of developmental signalling pathways and found that nearly all expected genes were present in each transcriptome. Our analyses show that both transcriptomes are of high quality. Lastly, we performed read mapping utilising our timed, stage specific RNA samples to identify differentially expressed contigs. The resources presented here will provide a firm basis for a variety of experimentation, both in developmental biology and in comparative genomic studies.

Genomics

Massively parallel whole-organism lineage tracing using CRISPR/Cas9 induced genetic scars

A key goal of developmental biology is to understand how a single cell transforms into a full-grown organism consisting of many cells. Although impressive progress has been made in lineage tracing using imaging approaches, analysis of vertebrate lineage trees has mostly been limited to relatively small subsets of cells. Here we present scartrace, a strategy for massively parallel clonal analysis based on Cas9 induced genetic scars in the zebrafish.

Systems Biology

Resurrected protein interaction networks reveal the strong rewiring that leads to network organisation after whole genome duplication

The evolution of plants is characterized by several rounds of ancient whole genome duplication, sometimes closely associated with the origin of large groups of species. A good example is the {gamma} triplication at the origin of core eudicots. Core eudicots comprise about 75% of flowering plants and are characterized by the canalization of reproductive development. To better understand the impact of this genomic event, we studied the protein interaction network of MADS-domain transcription factors, which are key regulators of reproductive development. We accurately inferred, resurrected and tested the interactions of ancestral proteins before and after the triplication and directly compared these ancestral networks to the networks of Arabidopsis and tomato. We find that the {gamma} triplication generated a dramatically innovated network that strongly rewired through the addition of many new interactions. Many of these interactions were established between paralogous proteins and a new interaction partner, establishing new redundancy. Simulations show that both node and edge addition through the triplication were important to maintain modularity in the network. In addition to generating insights into the impact of whole genome duplication and elementary processes involved in network evolution, our data provide a resource for comparative developmental biology in flowering plants.

Evolutionary Biology

The origin of animal multicellularity and cell differentiation

How animals evolved from their single-celled ancestors over 600 million years ago is poorly understood. Comparisons of genomes from animals and their closest relatives - choanoflagellates, filastereans and ichthyosporeans - have recently revealed the genomic landscape of animal origins. However, the cell and developmental biology of the first animals have been less well examined. Using principles from evolutionary cell biology, we reason that the last common ancestor of animals and choanoflagellates (the Urchoanozoan) used a collar complex - a flagellum surrounded by a microvillar collar - to capture bacterial prey. The origin of animal multicellularity likely occurred through the modification of pre-existing mechanisms for extracellular matrix synthesis and regulation of cytokinesis. The progenitors of animals likely developed clonally through serial division of flagellated cells, giving rise to sheets of cells that folded into spheres by a morphogenetic process comparable to that seen in modern choanoflagellate rosettes and calcareous sponge embryos. Finally, we infer that cell differentiation evolved in the animal stem-lineage by a combination of three mechanisms: division of labor from ancient plurifunctional cell types, conversion of temporally segregated phenotypes into spatially segregated cell types, and functional innovation.

evolutionary biology

Integrated transcriptome and epigenome analyses identify alternative splicing as a novel candidate linking histone modifications to embryonic stem cell fate decision

BackgroundUnderstanding the embryonic stem cell (ESC) fate decision between self-renewal and proper differentiation is important for developmental biology and regenerative medicine. Attention has focused on mechanisms involving histone modifications, alternative pre-mRNA splicing, and cell-cycle progression. However, their intricate interrelations and joint contributions to ESC fate decision remain unclear.\n\nResultsWe analyze the transcriptomes and epigenomes of human ESC and five types of differentiated cells. We identify thousands of alternatively spliced exons and reveal their development and lineage-dependent characterizations. Several histone modifications show dynamic changes in alternatively spliced exons and three are strongly associated with 52.8% of alternative splicing events upon hESC differentiation. The histone modification-associated alternatively spliced genes predominantly function in G2/M phases and ATM/ATR-mediated DNA damage response pathway for cell differentiation, whereas other alternatively spliced genes are enriched in the G1 phase and pathways for self-renewal. These results imply a potential epigenetic mechanism by which some histone modifications contribute to ESC fate decision through the regulation of alternative splicing in specific pathways and cell-cycle genes. Supported by experimental validations and extended dataset from Roadmap/ENCODE projects, we exemplify this mechanism by a cell cycle-related transcription factor, PBX1, which regulates the pluripotency regulatory network by binding to NANOG. We suggest that the isoform switch from PBX1a to PBX1b links H3K36me3 to hESC fate determination through the PSIP1/SRSF1 adaptor, which results in the exon skipping of PBX1.\n\nConclusionWe reveal the mechanism by which alternative splicing links histone modifications to stem cell fate decision.

bioinformatics

Independent erosion of conserved transcription factor binding sites points to shared hindlimb, vision, and scrotum loss in different mammals

Genetic variation in cis-regulatory elements is thought to be a major driving force in morphological and physiological change. However, identifying transcription factor binding events which code for complex traits remains a challenge, motivating novel means of detecting putatively important binding events. Using a curated set of 1,154 high-quality transcription factor motifs, we demonstrate that independently eroded binding sites are enriched for independently lost traits in three distinct pairs of placental mammals. We show that these independently eroded events pinpoint the loss of hindlimbs in dolphin and manatee, degradation of vision in naked mole-rat and star-nosed mole, and the loss of scrotum in white rhinoceros and Weddell seal. Our study exhibits a novel methodology to detect cis-regulatory mutations which help explain a portion of the molecular mechanism underlying complex trait formation and loss.\n\nAuthor SummaryEvolution has produced an astounding variety of species with incredibly diverse phenotypes. A central question in evolutionary developmental biology is how (and which) DNA evolves to encode all of these different traits. A prevailing hypothesis is that changes in regulatory DNA, short stretches of DNA which control the expression of protein-coding genes, drive important differences in trait formation between species. The basic building block of regulatory DNA is thought to be transcription factor binding sites, shortl genomic sequences which attract proteins whose central role is to control the rate of transcription. In this study, we asked whether the independent erosion of otherwise highly conserved transcription factor binding sites points to a trait shared between species which have undergone similar adaptations. We show that our method is able to point to the loss of hindlimbs in dolphin and manatee, poor vision in naked mole-rat and star-nosed mole, and loss of scrotum in Weddell seal and white rhinoceros. Overall, our study exhibits a means of detecting evolutionarily important genomic regions which help explain a portion of complex trait loss and retention.

evolutionary biology

Medaka population genome structure and demographic history unveiled via Genotyping-by-Sequencing

Medaka is a model organism in medicine, genetics, developmental biology and population genetics. Lab stocks composed of more than 100 local wild populations are available for research in these fields. Thus, medaka represents a potentially excellent bioresource for screening disease-risk- and adaptation-related genes in genome-wide association studies. Although the genetic population structure should be known before performing such an analysis, a comprehensive study on the genome-wide diversity of wild medaka populations has not been performed. Here, we performed genotyping-by-sequencing (GBS) for 81 and 12 medakas captured from a bioresource and the wild, respectively. Based on the GBS data, we evaluated the genetic population structure and estimated the demographic parameters using an approximate Bayesian computation (ABC) framework. The autosomal data confirmed that there were substantial differences between local populations and supported our previously proposed hypothesis on medaka dispersal based on mitochondrial genome (mtDNA) data. A new finding was that a local group that was thought to be a hybrid between the northern and the southern Japanese groups was actually a sister group of the northern Japanese group. Thus, this paper presents the first population-genomic study of medaka and reveals its population structure and history based on autosomal diversity.

evolutionary biology

Enhanced Waddington Landscape Model with Cell-CellCommunication Can Explain Molecular Mechanisms ofSelf-Organization

The molecular mechanisms of self-organization that orchestrate embryonic cells to create astonishing patterns have been among major questions of developmental biology. It is recently shown that embryonic stem cells (ESCs), when cultured on particular micropatterns, can self-organize and mimic early steps of pre-implantation embryogenesis. A systems-biology model to address this observation from a dynamical systems perspective is essential. Here, we propose a multicellular mathematical model for pattern formation during in vitro gastrulation of human ESCs. This model enhances the basic principles of Waddington epigenetic landscape with cell-cell communication, in order to enable pattern and tissue formation. To prevent overfitting of the model, there is a minimal number of parameters in the model, which are sufficient to address different experimental observations such as the formation of three germ layers and trophectoderm, responses to altered culture conditions and micropattern diameters, and unexpected spotted forms of the germ layers under certain conditions. This model provides a basis for in-silico modeling of self-organization.

systems biology

Experimentally Induced Metamorphosis in Axolotl (Ambystoma mexicanum) Under Constant Diet Restructures Microbiota Accompanied by Reduced Limb Regenerative Capacity

Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite life-long neoteny in nature and in captive-bred colonies, metamorphosis of these animals can be experimentally induced by administering Thyroid hormones (THs). However, biological consequences of this experimental procedure, such as host microbiota response and implications for regenerative capacity, remain largely unknown. Here, we systematically compared host bacterial microbiota associated with skin, stomach, gut tissues and fecal samples based on 16S rRNA gene sequences, along with limb regenerative capacity, between neotenic and metamorphic Axolotls. Our results show that distinct bacterial communities inhabit individual organs of Axolotl and undergo substantial restructuring through metamorphosis. Drastic restructuring was observed for skin microbiota, highlighted by a major transition from Firmicutes-enriched to Proteobacteria-enriched relative abundance and precipitously decreased diversity. Remarkably, shifts in microbiota was accompanied by a steep reduction in limb regenerative capacity. Fecal microbiota of neotenic and metamorphic Axolotl shared relatively higher similarity, suggesting that diet continues to shape microbiota despite fundamental transformations in the host digestive organs. The results provide novel insights into microbiological and regenerative aspects of Axolotl metamorphosis and will establish a baseline for future in-depth studies.

microbiology