Kaia Achim

High-throughput spatial mapping of single-cell RNA-seq data to tissue of origin

Understanding cell type identity in a multicellular organism requires the integration of gene expression profiles from individual cells with their spatial location in a particular tissue. Current technologies allow whole-transcriptome sequencing of spatially identified cells but lack the throughput needed to characterize complex tissues. Here we present a high-throughput method to identify the spatial origin of cells assayed by single-cell RNA-sequencing within a tissue of interest. Our approach is based on comparing complete, specificity-weighted mRNA profiles of a cell with positional gene expression profiles derived from a gene expression atlas. We show that this method allocates cells to precise locations in the brain of the marine annelid Platynereis dumerilii with a success rate of 81%. Our method is applicable to any system that has a reference gene expression database of sufficiently high resolution.

Identifying Cell Types from Spatially Referenced Single-Cell Expression Datasets

Complex tissues, such as the brain, are composed of multiple different cell types, each of which have distinct and important roles, for example in neural function. Moreover, it has recently been appreciated that the cells that make up these sub-cell types themselves harbour significant cell-to-cell heterogeneity, in particular at the level of gene expression. The ability to study this heterogeneity has been revolutionised by advances in experimental technology, such as Wholemount in Situ Hybridizations (WiSH) and single-cell RNA-sequencing. Consequently, it is now possible to study gene expression levels in thousands of cells from the same tissue type. After generating such data one of the key goals is to cluster the cells into groups that correspond to both known and putatively novel cell types. Whilst many clustering algorithms exist, they are typically unable to incorporate information about the spatial dependence between cells within the tissue under study. When such information exists it provides important insights that should be directly included in the clustering scheme. To this end we have developed a clustering method that uses a Hidden Markov Random Field (HMRF) model to exploit both quantitative measures of expression and spatial information. To accurately reflect the underlying biology, we extend current HMRF approaches by allowing the degree of spatial coherency to differ between clusters. We demonstrate the utility of our method using simulated data before applying it to cluster single cell gene expression data generated by applying WiSH to study expression patterns in the brain of the marine annelid Platynereis dumereilii. Our approach allows known cell types to be identified as well as revealing new, previously unexplored cell types within the brain of this important model system.

Structural evolution of cell types by step-wise assembly of cellular modules

Cell types are composed of cellular modules exerting specific subfunctions. The evolutionary emergence and diversification of these modules can be tracked through the comparative analysis of genomes. Here, we survey recent advances elucidating the origin of neurons, of smooth and striated muscle cells and of the T- and B-cells of the immune system in the diverging lineages of animal evolution. Gene presence and absence analyses in various metazoan genomes allow mapping the step-wise assembly of key modules - such as the postsynaptic density characteristic for neurons or the z-disk characteristic for striated muscle - on the animal evolutionary tree. Using this approach, first insight into the structural evolution of cell types can be gained.

Mechanisms regulating GABAergic neuron development

Neurons using gamma-aminobutyric acid (GABA) as their neurotransmitter are the main inhibitory neurons in the mature central nervous system (CNS) and show great variation in their form and function. GABAergic neurons are produced in all of the main domains of the CNS, where they develop from discrete regions of the neuroepithelium. Here, we review the gene expression and regulatory mechanisms controlling the main steps of GABAergic neuron development: early patterning of the proliferative neuroepithelium, production of postmitotic neural precursors, establishment of their identity and migration. By comparing the molecular regulation of these events across CNS, we broadly identify three regions utilizing distinct molecular toolkits for GABAergic fate determination: telencephalon–anterior diencephalon (DLX2 type), posterior diencephalon–midbrain (GATA2 type) and hindbrain–spinal cord (PTF1A and TAL1 types). Similarities and differences in the molecular regulatory mechanisms reveal the core determinants of a GABAergic neuron as well as provide insights into generation of the vast diversity of these neurons.

Whole-organism cellular gene-expression atlas reveals conserved cell types in the ventral nerve cord of Platynereis dumerilii

The comparative study of cell types is a powerful approach toward deciphering animal evolution. To avoid selection biases, however, comparisons ideally involve all cell types present in a multicellular organism. Here, we use image registration and a newly developed “Profiling by Signal Probability Mapping” algorithm to generate a cellular resolution 3D expression atlas for an entire animal. We investigate three-segmented young worms of the marine annelid Platynereis dumerilii, with a rich diversity of differentiated cells present in relatively low number. Starting from whole-mount expression images for close to 100 neural specification and differentiation genes, our atlas identifies and molecularly characterizes 605 bilateral pairs of neurons at specific locations in the ventral nerve cord. Among these pairs, we identify sets of neurons expressing similar combinations of transcription factors, located at spatially coherent anterior-posterior, dorsal-ventral, and medial-lateral coordinates that we interpret as cell types. Comparison with motor and interneuron types in the vertebrate neural tube indicates conserved combinations, for example, of cell types cospecified by Gata1/2/3 and Tal transcription factors. These include V2b interneurons and the central spinal fluid-contacting Kolmer-Agduhr cells in the vertebrates, and several neuron types in the intermediate ventral ganglionic mass in the annelid. We propose that Kolmer-Agduhr cell-like mechanosensory neurons formed part of the mucociliary sole in protostome-deuterostome ancestors and diversified independently into several neuron types in annelid and vertebrate descendants.

Gata2 and Gata3 regulate the differentiation of serotonergic and glutamatergic neuron subtypes of the dorsal raphe

Serotonergic and glutamatergic neurons of the dorsal raphe regulate many brain functions and are important for mental health. Their functional diversity is based on molecularly distinct subtypes; however, the development of this heterogeneity is poorly understood. We show that the ventral neuroepithelium of mouse anterior hindbrain is divided into specific subdomains giving rise to serotonergic neurons as well as other types of neurons and glia. The newly born serotonergic precursors are segregated into distinct subpopulations expressing vesicular glutamate transporter 3 (Vglut3) or serotonin transporter (Sert). These populations differ in their requirements for transcription factors Gata2 and Gata3, which are activated in the post-mitotic precursors. Gata2 operates upstream of Gata3 as a cell fate selector in both populations, whereas Gata3 is important for the differentiation of the Sert+ precursors and for the serotonergic identity of the Vglut3+ precursors. Similar to the serotonergic neurons, the Vglut3-expressing glutamatergic neurons, located in the central dorsal raphe, are derived from neural progenitors in the ventral hindbrain and express Pet1. Furthermore, both Gata2 and Gata3 are redundantly required for their differentiation. Our study demonstrates lineage relationships of the dorsal raphe neurons and suggests that functionally significant heterogeneity of these neurons is established early during their differentiation. Summary: Dorsal raphe neurons in mice exhibit functionally significant heterogeneity that is established early during their differentiation via the actions of Gata2 and Gata3.

Whole-body single-cell sequencing reveals transcriptional domains in the annelid larval body.

Abstract Animal bodies comprise diverse arrays of cells. To characterize cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii. We identify five transcriptionally distinct groups of differentiated cells, each expressing a unique set of transcription factors and effector genes that implement cellular phenotypes. Spatial mapping of cells into a cellular expression atlas, and wholemount in situ hybridization of group‐specific genes reveals spatially coherent transcriptional domains in the larval body, comprising, for example, apical sensory‐neurosecretory cells versus neural/epidermal surface cells. These domains represent new, basic subdivisions of the annelid body based entirely on differential gene expression, and are composed of multiple, transcriptionally similar cell types. They do not represent clonal domains, as revealed by developmental lineage analysis. We propose that the transcriptional domains that subdivide the annelid larval body represent families of related cell types that have arisen by evolutionary diversification. Their possible evolutionary conservation makes them a promising tool for evo‐devo research.

Whole-body single-cell sequencing of the Platynereis larva reveals a subdivision into apical versus non-apical tissues

Animal bodies comprise a diverse array of tissues and cells. To characterise cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii1–4. We identify five transcriptionally distinct groups of differentiated cells that are spatially coherent, as revealed by spatial mapping5. Besides somatic musculature, ciliary bands and midgut, we find a group of cells located at the apical tip of the animal, comprising sensory-peptidergic neurons, and another group composed of non-apical neural and epidermal cells covering the rest of the body. These data establish a basic subdivision of the larval body surface into molecularly defined apical versus non-apical tissues, and support the evolutionary conservation of the apical nervous system as a distinct part of the bilaterian brain6.

Single Cell Genomics meeting in Stockholm: from single cells to cell types

A report on the second Single Cell Genomics conference held in Stockholm, Sweden, September 9-11, 2014.

From spiral cleavage to bilateral symmetry: The developmental cell lineage of the annelid brain

The spiral cleavage pattern is characteristic for Spiralia (Lophotrochozoa), a large assembly of marine invertebrates. In most cases, spiral cleavage produces freely swimming, trochophora-type larvae with a simple nervous system that controls ciliary locomotion. These larvae acquire bilateral symmetry, as manifested for example in the larval brain. The transition from the rotational symmetry of spiral cleavage into the bilateral adult body has not yet been understood. Here, we present the developmental cell lineage of the brain of the annelid Platynereis dumerilii from the zygote until the mid-trochophore stage (~30 hpf), in combination with a gene expression atlas for several embryonic and larval stages. Comparison of multiple embryos reveals a highly stereotypical development and an invariant cell lineage of the differentiated cell types. In addition, we observe a fundamental subdivision of the larval brain into a highly proliferative dorsolateral region and an early differentiating ventromedial region that gives rise to the apical nervous system. The transition from rotational to bilateral symmetry progresses gradually from the lateral to the central regions. Strikingly, the spiral-to-bilateral transition does not involve extensive cell migration. Rather, corresponding cells in different spiral quadrants acquire highly divergent identities in line with their bilateral position.