The cellular landscape of the endochondral bone during the transition to extrauterine life.

The cellular complexity of the endochondral bone underlies its essential and pleiotropic roles during organismal life. While the adult bone has received significant attention, we still lack a deep understanding of the perinatal bone cellulome. Here, we have profiled the full composition of the murine endochondral bone at the single-cell level during the transition from fetal to newborn life and in comparison with the adult tissue, with particular emphasis on the mesenchymal compartment. The perinatal bone contains different fibroblastic clusters with blastema-like characteristics in organizing and supporting skeletogenesis, angiogenesis and hematopoiesis. Our data also suggest dynamic inter- and intra-compartment interactions, as well as a bone marrow milieu that seems prone to anti-inflammation, which we hypothesize is necessary to ensure the proper program of lymphopoiesis and the establishment of central and peripheral tolerance in early life. Our study provides an integrative roadmap for the future design of genetic and cellular functional assays to validate cellular interactions and lineage relationships within the perinatal bone.

CeLaVi: an interactive cell lineage visualization tool.

Recent innovations in genetics and imaging are providing the means to reconstruct cell lineages, either by tracking cell divisions using live microscopy, or by deducing the history of cells using molecular recorders. A cell lineage on its own, however, is simply a description of cell divisions as branching events. A major goal of current research is to integrate this description of cell relationships with information about the spatial distribution and identities of the cells those divisions produce. Visualizing, interpreting and exploring these complex data in an intuitive manner requires the development of new tools. Here we present CeLaVi, a web-based visualization tool that allows users to navigate and interact with a representation of cell lineages, whilst simultaneously visualizing the spatial distribution, identities and properties of cells. CeLaVi's principal functions include the ability to explore and manipulate the cell lineage tree; to visualise the spatial distribution of cell clones at different depths of the tree; to colour cells in the 3D viewer based on lineage relationships; to visualise various cell qualities on the 3D viewer (e.g. gene expression, cell type) and to annotate selected cells/clones. All these capabilities are demonstrated with four different example data sets. CeLaVi is available at http://www.celavi.pro.

Mapping Selection within Drosophila melanogaster Embryo's Anatomy.

We present a survey of selection across Drosophila melanogaster embryonic anatomy. Our approach integrates genomic variation, spatial gene expression patterns, and development with the aim of mapping adaptation over the entire embryo's anatomy. Our adaptation map is based on analyzing spatial gene expression information for 5,969 genes (from text-based annotations of in situ hybridization data directly from the BDGP database, Tomancak et al. 2007) and the polymorphism and divergence in these genes (from the project DGRP, Mackay et al. 2012).The proportion of nonsynonymous substitutions that are adaptive, neutral, or slightly deleterious are estimated for the set of genes expressed in each embryonic anatomical structure using the distribution of fitness effects-alpha method (Eyre-Walker and Keightley 2009). This method is a robust derivative of the McDonald and Kreitman test (McDonald and Kreitman 1991). We also explore whether different anatomical structures differ in the phylogenetic age, codon usage, or expression bias of the genes they express and whether genes expressed in many anatomical structures show more adaptive substitutions than other genes.We found that: 1) most of the digestive system and ectoderm-derived structures are under selective constraint, 2) the germ line and some specific mesoderm-derived structures show high rates of adaptive substitution, and 3) the genes that are expressed in a small number of anatomical structures show higher expression bias, lower phylogenetic ages, and less constraint.

How complexity increases in development: An analysis of the spatial-temporal dynamics of 1218 genes in Drosophila melanogaster.

One of the most apparent phenomena in development is that it starts with something apparently simple and leads to something clearly complex with a specific and functional structure. At the level of gene expression it seems also clear that the embryo becomes progressively compartmentalized over time and space. However, there have not been any systematic attempts to quantify how this occurs. Here, we present a quantitative analysis of the compartmentalization and spatial complexity of gene expression in Drosophila melanogaster over developmental time by analyzing thousands of gene expression spatial patterns from FlyExpress database. We use three different mathematical measures of compartmentalization of gene expression in space. All these measures show a similar non-linear increase in compartmentalization over time, with the most dramatic change occurring from the maternal to the early gastrula stage. Transcription factors and growth factors showed an earlier compartmentalization. Finally, we partitioned the embryo space in 257 equally sized regions and clustered them depending on their expression similarity, within and between stages. This provides a global overview about the effective degree of differentiation and compartmentalization between body parts at each developmental stage and when and where in the embryo there are more changes, due to signaling or movement.

Single-cell atlases of two lophotrochozoan larvae highlight their complex evolutionary histories.

Pelagic larval stages are widespread across animals, yet it is unclear whether larvae were present in the last common ancestor of animals or whether they evolved multiple times due to common selective pressures. Many marine larvae are at least superficially similar; they are small, swim through the beating of bands of cilia, and sense the environment with an apical organ. To understand these similarities, we have generated single-cell atlases for marine larvae from two animal phyla and have compared their cell types. We found clear similarities among ciliary band cells and between neurons of the apical organ in the two larvae pointing to possible homology of these structures, suggesting a single origin of larvae within Spiralia. We also find several clade-specific innovations in each larva, including distinct myocytes and shell gland cells in the oyster larva. Oyster shell gland cells express many recently evolved genes that have made previous gene age estimates for the origin of trochophore larvae too young.

Benchmarked approaches for reconstruction of in vitro cell lineages and in silico models of C. elegans and M. musculus developmental trees.

The recent advent of CRISPR and other molecular tools enabled the reconstruction of cell lineages based on induced DNA mutations and promises to solve the ones of more complex organisms. To date, no lineage reconstruction algorithms have been rigorously examined for their performance and robustness across dataset types and number of cells. To benchmark such methods, we decided to organize a DREAM challenge using in vitro experimental intMEMOIR recordings and in silico data for a C. elegans lineage tree of about 1,000 cells and a Mus musculus tree of 10,000 cells. Some of the 22 approaches submitted had excellent performance, but structural features of the trees prevented optimal reconstructions. Using smaller sub-trees as training sets proved to be a good approach for tuning algorithms to reconstruct larger trees. The simulation and reconstruction methods here generated delineate a potential way forward for solving larger cell lineage trees such as in mouse.

Is it possible to reconstruct an accurate cell lineage using CRISPR recorders?

Cell lineages provide the framework for understanding how cell fates are decided during development. Describing cell lineages in most organisms is challenging; even a fruit fly larva has ~50,000 cells and a small mammal has >1 billion cells. Recently, the idea of applying CRISPR to induce mutations during development, to be used as heritable markers for lineage reconstruction, has been proposed by several groups. While an attractive idea, its practical value depends on the accuracy of the cell lineages that can be generated. Here, we use computer simulations to estimate the performance of these approaches under different conditions. We incorporate empirical data on CRISPR-induced mutation frequencies in Drosophila. We show significant impacts from multiple biological and technical parameters - variable cell division rates, skewed mutational outcomes, target dropouts and different sequencing strategies. Our approach reveals the limitations of published CRISPR recorders, and indicates how future implementations can be optimised. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Computational discovery of hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity Numbers.

In some eukaryotes, a 'hidden break' has been described in which the 28S ribosomal RNA molecule is cleaved into two subparts. The break is common in protostome animals (arthropods, molluscs, annelids etc.), but a break has also been reported in some vertebrates and non-metazoan eukaryotes. We present a new computational approach to determine the presence of the hidden break in 28S rRNAs using mapping of RNA-Seq data. We find a homologous break is present across protostomes although it has been lost in a small number of taxa. We show that rare breaks in vertebrate 28S rRNAs are not homologous to the protostome break. A break is found in just 4 out of 331 species of non-animal eukaryotes studied and, in three of these, the break is located in the same position as the protostome break suggesting a striking instance of convergent evolution. RNA Integrity Numbers (RIN) rely on intact 28S rRNA and will be consistently underestimated in the great majority of animal species with a break.

Adaptation and Conservation throughout the Drosophila melanogaster Life-Cycle.

Previous studies of the evolution of genes expressed at different life-cycle stages of Drosophila melanogaster have not been able to disentangle adaptive from nonadaptive substitutions when using nonsynonymous sites. Here, we overcome this limitation by combining whole-genome polymorphism data from D. melanogaster and divergence data between D. melanogaster and Drosophila yakuba. For the set of genes expressed at different life-cycle stages of D. melanogaster, as reported in modENCODE, we estimate the ratio of substitutions relative to polymorphism between nonsynonymous and synonymous sites (α) and then α is discomposed into the ratio of adaptive (ωa) and nonadaptive (ωna) substitutions to synonymous substitutions. We find that the genes expressed in mid- and late-embryonic development are the most conserved, whereas those expressed in early development and postembryonic stages are the least conserved. Importantly, we found that low conservation in early development is due to high rates of nonadaptive substitutions (high ωna), whereas in postembryonic stages it is due, instead, to high rates of adaptive substitutions (high ωa). By using estimates of different genomic features (codon bias, average intron length, exon number, recombination rate, among others), we also find that genes expressed in mid- and late-embryonic development show the most complex architecture: they are larger, have more exons, more transcripts, and longer introns. In addition, these genes are broadly expressed among all stages. We suggest that all these genomic features are related to the conservation of mid- and late-embryonic development. Globally, our study supports the hourglass pattern of conservation and adaptation over the life-cycle.

How complexity increases in development: An analysis of the spatial-temporal dynamics of Gene expression in Ciona intestinalis.

The increase in complexity in an embryo over developmental time is perhaps one of the most intuitive processes of animal development. It is also intuitive that the embryo becomes progressively compartmentalized over time and space. In spite of this intuitiveness, there are no systematic attempts to quantify how this occurs. Here, we present a quantitative analysis of the compartmentalization and spatial complexity of Ciona intestinalis over developmental time by analyzing thousands of gene expression spatial patterns from the ANISEED database. We measure compartmentalization in two ways: as the relative volume of expression of genes and as the disparity in gene expression between body parts. We also use a measure of the curvature of each gene expression pattern in 3D space. These measures show a similar increase over time, with the most dramatic change occurring from the 112-cell stage to the early tailbud stage. Combined, these measures point to a global pattern of increase in complexity in the Ciona embryo. Finally, we cluster the different regions of the embryo depending on their gene expression similarity, within and between stages. Results from this clustering analysis, which partially correspond to known fate maps, provide a global quantitative overview about differentiation and compartmentalization between body parts at each developmental stage.