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NEWS & VIEWS RESEARCH unaffected. Remarkably, forced expression whether calsyntenin 3β functions as a general Maryland 21218, USA. of S100b in the brown adipocytes of Clstn3b­ ER chaperone for the secretion of proteins that e-mail: [email protected] knockout mice is sufficient to correct the deficits lack signal peptides, or is specific for S100b. 1. Cannon, B. & Nedergaard, J. Physiol. Rev. 84, in sympathetic innervation and thermogenesis In humans, BAT had been thought to 277–359 (2004). in these animals. The emerging picture is of an exist only in infants, until imaging studies 2. Zeng, X. et al. Nature 569, 229–235 (2019). adipocyte­derived mechanism that mediates revealed deposits of thermogenic brown fat 3. Bartness, T. J., Vaughan, C. H. & Song, C. K. Int. J. crosstalk between brown adipocytes and in adults10. Clstn3b is expressed in human Obesity 34, S36–S42 (2010). 4. Wang, W. & Seale, P. Nature Rev. Mol. Cell Biol. 17, innervating sympathetic fibres (Fig. 1). adipose tissue, and might be involved in the 691–702 (2016). Zeng and colleagues’ findings raise several regulation of BAT innervation, as it is in mice. 5. Ludwin, S. K., Kosek, J. C. & Eng, L. F. questions. For example, how is calsyntenin 3β Zeng and colleagues’ study might therefore J. Comp. Neurol. 165, 197–207 (1976). 6. Duteil, D. et al. Cell Rep. 17, 1008–1021 (2016). expression regulated in brown adipocytes? The inform therapeutic strategies to enhance 7. Glebova, N. O. & Ginty, D. D. J. Neurosci. 24, authors found that one upstream regulator sympathetic innervation, and thereby harness 743–751 (2004). of this protein is the enzyme lysine­specific the thermogenic potential of BAT to combat 8. Hall, J. A. et al. Endocrinology 151, 4573–4582 (2010). demethylase 1 (LSD1), which mediates obesity and its metabolic consequences. ■ 9. Rabouille, C. Trends Cell Biol. 27, 230–240 (2017). 10. Nedergaard, J., Bengtsson, T. & Cannon, B. Am. J. the differentiation of brown and beige fat. Physiol. Endocrinol. Metab. 293, E444–E452 (2007). Adipose­specific loss of LSD1 in mice causes Rejji Kuruvilla is in the Department of downregulation of BAT­specific genes and Biology, Johns Hopkins University, Baltimore, This article was published online on 1 May 2019. aberrant induction of genes typically found in white adipose tissue6. The growth of sympathetic neurons mostly TRANSCRIPTION relies on a protein called nerve growth factor (NGF). NGF­deficient mice have pronounced disruption of sympathetic innervation of multi ple peripheral tissues7, but BAT Gene-expression maps innervation has not been specifically examined in this context. It remains to be seen whether NGF acts together with S100b to coordinate fall into place BAT innervation, or is dispensable, as in the innervation of the trachea7. Could other Knowing the gene-expression pattern of individual cells can unlock their factors derived from peripheral tissue control identity. A refined method for generating cellular RNA profiles offers a way the sympathetic innervation of specific to obtain such data at a high level of spatial resolution in intact tissues. targets? If so, they could be useful tools for manipulating such innervation and studying its role in individual tissues. SAMANTHA A. MORRIS has brought huge efficiency gains and cost Sympathetic innervation controls the reductions3–5. However, single­cell RNA expression of genes involved in thermo­ onitoring messenger RNA in cells sequencing requires tissue disruption and cell genesis and the differentiation of brown and is a way to gather data that provides destruction, which causes loss of spatial infor­ beige fat4. However, Zeng et al. found that many biological insights. Improve­ mation about cellular location that would be loss of Clstn3b does not affect the expression Mments in the methods for detecting cellular valuable for cell­type identification. of genes involved in adipocyte development, mRNA are helping to provide more­detailed Efforts to retain spatial information are a thermogenesis or mitochondrial function, pictures of gene expression in the cells of a current focus in the development of methods including the gene that encodes UCP1. Only particular tissue. Writing in Science, Rodriques for single­cell analysis. For example, a the expression of the Dio2 gene, which encodes et al.1 describe a new approach to assessing method called multiplexed in situ hybridiza­ an enzyme that produces the active form of a cellular RNA that they term Slide­seq. This tion, along with certain sequencing techniques, thyroid hormone, is significantly altered by the versatile technique couples high­throughput has enabled RNA to be monitored on a sub­ loss or over expression of Clstn3b. The Dio2 RNA sequencing with a way to capture spatial cellular scale in intact tissues6–8. However, protein is abundant in BAT, and, as is seen in information about the location of the analysed these approaches require substantial technical the Clstn3b­knockout mice, mice lacking Dio2 cells in tissues. expertise, which has limited their widespread have impaired thermogenesis and decreased Scientists often try to tackle the complexity adoption. cold tolerance, and are susceptible to diet­ of a biological system by breaking it down to Rodriques and colleagues’ approach is induced obesity8. Altered thyroid signalling focus on cells, and cataloguing these individual conceptually rooted in an earlier3 method for might therefore contribute to BAT dysfunction units on the basis of their identity. Conven­ single­cell RNA analysis. In that technique, in the absence of calsyntenin 3β. tional microscopic imaging of cells in a tissue called Drop­seq, the tissue being analysed The final issue arising from Zeng and can provide much information, such as cell is disrupted to separate the cells, which are colleagues’ work is how calsyntenin 3β type and function, but imaging data of many then loaded onto a microfluidic device. Indi­ promotes the secretion of S100b. The synthesis cells can still lack a high level of detail about vidual cells are encapsulated in nanolitre­ of proteins destined for cellular secretion cellular features — the characteristics known scale droplets together with a microbead is initiated in the cytoplasm. The nascent as the cellular phenotype. coated with copies of a DNA sequence that proteins typically contain an amino­acid The development of techniques for provides a unique ‘barcode’ for the identifi­ sequence known as a signal peptide, which rigorously analysing the molecular contents cation of material from each droplet (which directs them from the cytoplasm to the ER of individual cells offers an alternative way to is assumed to contain a single cell). The for the start of their secretory journey while capture thousands of cellular features and gen­ cells are broken open, which allows the cel­ they are still being synthesized. However, some erate an unbiased picture of the cells in a given lular RNA to bind to the DNA barcodes. A secreted proteins lack signal peptides and tissue. For example, single­cell RNA sequen­ step called reverse transcription generates are imported to the ER after they have been cing has been rapidly and widely adopted since DNA corresponding to the captured RNA synthesized9. S100b lacks a peptide signal, its emergence ten years ago2. The use of micro­ sequences, and this DNA is tagged with the and so the new findings raise the question of fluidic­based technologies in this approach barcode DNA. DNA sequencing then enables ©2019 Spri nger Nature Li mited. All ri ghts reserved. ©2019 Spri nger Nature Li mited. All ri ghts reserved. 9 MAY 2019 | VOL 569 | NATURE | 197 RESEARCH NEWS & VIEWS this region by analysing 66 sequential tissue a Glass Microbead b c sections. The authors generated RNA profiles slide layer Frozen tissue of 1.5 million microbeads, demonstrating that Slide­seq can be scaled up as necessary. Their method identified gene­expression patterns that could be used to assign distinct cerebel­ lar cell types together into spatially defined subpopulations in the mouse brain. Such Location of RNA expression of many genes is spatially depend­ Microbead sequences assigned ent and is independent of cell type, but this valuable information is usually lost in conven­ tional single­cell profiling approaches during the cell­dissociation step9. The authors also Unique DNA characterized cellular transcription dynamics ‘barcode’ RNA sequence in a mouse model of traumatic brain injury, DNA Reverse revealing that an initial wave of cell prolif­ sequencing transcription eration after injury was followed by cellular Spatially resolved RNA prole of tissue differentiation. One limitation of Slide­seq is that infor­ mation about the size and shape of each cell Barcode-tagged and other key physical properties is not deter­ DNA mined, unlike the case for RNA­profiling methods that use a microscopy technique 10 Barcode location called histology. A Nature paper published recorded Microbeads dissociated in March offers a way to capture such cellular and DNA sequenced features while also tracking the subcellular localization of mRNA — this technique to Figure 1 | Recording the spatial location of RNA sequences in tissues. Rodriques et al.1 report a image RNA transcripts in cells in tissues is technique called Slide­seq, which enables RNA in frozen tissue samples to be observed in a way that called seqFISH+. Previous approaches similar provides spatial information about gene­expression patterns. a, The first step is the preparation of to seqFISH+ were restricted by the optical­ glass slides that contain a layer of microbeads. Each microbead has copies of a unique DNA sequence resolution limits of microscopes and the attached that provides an identification ‘barcode’ (only one barcode shown per bead). Sequencing high density of mRNA transcripts in a cell. these barcodes directly on the slide allowed the authors to record the slide position associated with each barcode. b, Rodriques and colleagues placed a frozen sample of tissue, such as a sample of mouse SeqFISH+ overcame these problems through brain, on the slide.

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