Raju Tomer

Advanced CLARITY for rapid and high-resolution imaging of intact tissues

CLARITY is a method for chemical transformation of intact biological tissues into a hydrogel-tissue hybrid, which becomes amenable to interrogation with light and macromolecular labels while retaining fine structure and native biological molecules. This emerging accessibility of information from large intact samples has created both new opportunities and new challenges. Here we describe protocols spanning multiple dimensions of the CLARITY workflow, ranging from simple, reliable and efficient lipid removal without electrophoretic instrumentation (passive CLARITY) to optimized objectives and integration with light-sheet optics (CLARITY-optimized light-sheet microscopy (COLM)) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution. The entire protocol takes from 7–28 d to complete for an adult mouse brain, including hydrogel embedding, full lipid removal, whole-brain antibody staining (which, if needed, accounts for 7–10 of the days), and whole-brain high-resolution imaging; timing within this window depends on the choice of lipid removal options, on the size of the tissue, and on the number and type of immunostaining rounds performed. This protocol has been successfully applied to the study of adult mouse, adult zebrafish and adult human brains, and it may find many other applications in the structural and molecular analysis of large assembled biological systems.

Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy

Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo. SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.

Ancient animal microRNAs and the evolution of tissue identity

The spectacular escalation in complexity in early bilaterian evolution correlates with a strong increase in the number of microRNAs. To explore the link between the birth of ancient microRNAs and body plan evolution, we set out to determine the ancient sites of activity of conserved bilaterian microRNA families in a comparative approach. We reason that any specific localization shared between protostomes and deuterostomes (the two major superphyla of bilaterian animals) should probably reflect an ancient specificity of that microRNA in their last common ancestor. Here, we investigate the expression of conserved bilaterian microRNAs in Platynereis dumerilii, a protostome retaining ancestral bilaterian features, in Capitella, another marine annelid, in the sea urchin Strongylocentrotus, a deuterostome, and in sea anemone Nematostella, representing an outgroup to the bilaterians. Our comparative data indicate that the oldest known animal microRNA, miR-100, and the related miR-125 and let-7 were initially active in neurosecretory cells located around the mouth. Other sets of ancient microRNAs were first present in locomotor ciliated cells, specific brain centres, or, more broadly, one of four major organ systems: central nervous system, sensory tissue, musculature and gut. These findings reveal that microRNA evolution and the establishment of tissue identities were closely coupled in bilaterian evolution. Also, they outline a minimum set of cell types and tissues that existed in the protostome–deuterostome ancestor.

Intact-Brain Analyses Reveal Distinct Information Carried by SNc Dopamine Subcircuits

Recent progress in understanding the diversity of midbrain dopamine neurons has highlighted the importance--and the challenges--of defining mammalian neuronal cell types. Although neurons may be best categorized using inclusive criteria spanning biophysical properties, wiring of inputs, wiring of outputs, and activity during behavior, linking all of these measurements to cell types within the intact brains of living mammals has been difficult. Here, using an array of intact-brain circuit interrogation tools, including CLARITY, COLM, optogenetics, viral tracing, and fiber photometry, we explore the diversity of dopamine neurons within the substantia nigra pars compacta (SNc). We identify two parallel nigrostriatal dopamine neuron subpopulations differing in biophysical properties, input wiring, output wiring to dorsomedial striatum (DMS) versus dorsolateral striatum (DLS), and natural activity patterns during free behavior. Our results reveal independently operating nigrostriatal information streams, with implications for understanding the logic of dopaminergic feedback circuits and the diversity of mammalian neuronal cell types.

A small non‐coding RNA of the invasion gene island (SPI‐1) represses outer membrane protein synthesis from the Salmonella core genome

The Salmonella pathogenicity island (SPI‐1) encodes ∼35 proteins involved in assembly of a type III secretion system (T3SS) which endows Salmonella with the ability to invade eukaryotic cells. We have discovered a novel SPI‐1 gene, invR, which expresses an abundant small non‐coding RNA (sRNA). The invR gene, which we identified in a global search for new Salmonella sRNA genes, is activated by the major SPI‐1 transcription factor, HilD, under conditions that favour host cell invasion. The RNA chaperone, Hfq, is essential for the in vivo stability of the ∼80 nt InvR RNA. Hfq binds InvR with high affinity in vitro, and InvR co‐immunoprecipitates with FLAG epitope‐tagged Hfq in Salmonella extracts. Surprisingly, deletion/overexpression of invR revealed no phenotype in SPI‐1 regulation. In contrast, we find that InvR represses the synthesis of the abundant OmpD porin encoded by the Salmonella core genome. As invR is conserved in the early branching Salmonella bongori, we speculate that porin repression by InvR may have aided successful establishment of the SPI‐1 T3SS after horizontal acquisition in the Salmonella lineage. This study identifies the first regulatory RNA of an enterobacterial pathogenicity island, and new roles for Hfq and HilD in SPI‐1 gene expression.

Larval body patterning and apical organs are conserved in animal evolution

BackgroundPlanktonic ciliated larvae are characteristic for the life cycle of marine invertebrates. Their most prominent feature is the apical organ harboring sensory cells and neurons of largely undetermined function. An elucidation of the relationships between various forms of primary larvae and apical organs is key to understanding the evolution of animal life cycles. These relationships have remained enigmatic due to the scarcity of comparative molecular data.ResultsTo compare apical organs and larval body patterning, we have studied regionalization of the episphere, the upper hemisphere of the trochophore larva of the marine annelid Platynereis dumerilii. We examined the spatial distribution of transcription factors and of Wnt signaling components previously implicated in anterior neural development. Pharmacological activation of Wnt signaling with Gsk3β antagonists abolishes expression of apical markers, consistent with a repressive role of Wnt signaling in the specification of apical tissue. We refer to this Wnt-sensitive, six3- and foxq2-expressing part of the episphere as the ‘apical plate’. We also unraveled a molecular signature of the apical organ - devoid of six3 but expressing foxj, irx, nkx3 and hox - that is shared with other marine phyla including cnidarians. Finally, we characterized the cell types that form part of the apical organ by profiling by image registration, which allows parallel expression profiling of multiple cells. Besides the hox-expressing apical tuft cells, this revealed the presence of putative light- and mechanosensory as well as multiple peptidergic cell types that we compared to apical organ cell types of other animal phyla.ConclusionsThe similar formation of a six3+, foxq2+ apical plate, sensitive to Wnt activity and with an apical tuft in its six3-free center, is most parsimoniously explained by evolutionary conservation. We propose that a simple apical organ - comprising an apical tuft and a basal plexus innervated by sensory-neurosecretory apical plate cells - was present in the last common ancestors of cnidarians and bilaterians. One of its ancient functions would have been the control of metamorphosis. Various types of apical plate cells would then have subsequently been added to the apical organ in the divergent bilaterian lineages. Our findings support an ancient and common origin of primary ciliated larvae.

SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function.

The goal of understanding living nervous systems has driven interest in high-speed and large field-of-view volumetric imaging at cellular resolution. Light sheet microscopy approaches have emerged for cellular-resolution functional brain imaging in small organisms such as larval zebrafish, but remain fundamentally limited in speed. Here, we have developed SPED light sheet microscopy, which combines large volumetric field-of-view via an extended depth of field with the optical sectioning of light sheet microscopy, thereby eliminating the need to physically scan detection objectives for volumetric imaging. SPED enables scanning of thousands of volumes-per-second, limited only by camera acquisition rate, through the harnessing of optical mechanisms that normally result in unwanted spherical aberrations. We demonstrate capabilities of SPED microscopy by performing fast sub-cellular resolution imaging of CLARITY mouse brains and cellular-resolution volumetric Ca(2+) imaging of entire zebrafish nervous systems. Together, SPED light sheet methods enable high-speed cellular-resolution volumetric mapping of biological system structure and function.

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

Development of the annelid axochord: Insights into notochord evolution

Origin of the spine lies in a worm The notochord, the developmental backbone precursor, defines chordates—the group of animals to which humans belong. The origin of the notochord remains mysterious. Lauri et al. report the identification of a longitudinal muscle in an annelid worm that displays striking similarities to the notochord regarding position, developmental origin, and expression profile. Similar muscles, termed axochords, are found in various invertebrate phyla. These data suggest that the last common ancestor of bilaterians already possessed contractile midline tissue that, via stiffening, developed into a cartilaginous rod in the chordate line. Science, this issue p. 1365 A comparative study suggests that the chordate notochord evolved from a ventral midline muscle in bilaterian ancestors. The origin of chordates has been debated for more than a century, with one key issue being the emergence of the notochord. In vertebrates, the notochord develops by convergence and extension of the chordamesoderm, a population of midline cells of unique molecular identity. We identify a population of mesodermal cells in a developing invertebrate, the marine annelid Platynereis dumerilii, that converges and extends toward the midline and expresses a notochord-specific combination of genes. These cells differentiate into a longitudinal muscle, the axochord, that is positioned between central nervous system and axial blood vessel and secretes a strong collagenous extracellular matrix. Ancestral state reconstruction suggests that contractile mesodermal midline cells existed in bilaterian ancestors. We propose that these cells, via vacuolization and stiffening, gave rise to the chordate notochord.

Shedding light on the system: studying embryonic development with light sheet microscopy

Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for the life sciences. LSFM provides an exceptionally high imaging speed, high signal-to-noise ratio, low level of photo-bleaching and good optical penetration depth. This unique combination of capabilities makes light sheet-based microscopes highly suitable for live imaging applications. There is an outstanding potential in applying this technology to the quantitative study of embryonic development. Here, we provide an overview of the different basic implementations of LSFM, review recent technical advances in the field and highlight applications in the context of embryonic development. We conclude with a discussion of promising future directions.