Vikas Trivedi

Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging

Time-lapse imaging of multiple labels is challenging for biological imaging as noise, photobleaching and phototoxicity compromise signal quality, while throughput can be limited by processing time. Here, we report software called Hyper-Spectral Phasors (HySP) for denoising and unmixing multiple spectrally overlapping fluorophores in a low signal-to-noise regime with fast analysis. We show that HySP enables unmixing of seven signals in time-lapse imaging of living zebrafish embryos.

Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy

Significance Most environments host a poorly understood microbial diversity. In recent years, work on gene inventories and metagenomics has revealed much about the microbial species and metabolic genes that may be present in situ; however, connecting microbial species with environmental function has lagged. Here a combination of emerging single-cell and other approaches revealed the gut microbes that may catalyze a key activity in their termite hosts. The results implicate a previously unappreciated deltaproteobacterium living on a gut protist. Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a “lower,” wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based gene–species associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche.

Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration

We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines.

Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids

The establishment of the anteroposterior (AP) axis is a crucial step during animal embryo development. In mammals, genetic studies have shown that this process relies on signals spatiotemporally deployed in the extra-embryonic tissues that locate the position of the head and the onset of gastrulation, marked by T/Brachyury (T/Bra) at the posterior of the embryo. Here, we use gastruloids, mESC-based organoids, as a model system with which to study this process. We find that gastruloids localise T/Bra expression to one end and undergo elongation similar to the posterior region of the embryo, suggesting that they develop an AP axis. This process relies on precisely timed interactions between Wnt/β-catenin and Nodal signalling, whereas BMP signalling is dispensable. Additionally, polarised T/Bra expression occurs in the absence of extra-embryonic tissues or localised sources of signals. We suggest that the role of extra-embryonic tissues in the mammalian embryo might not be to induce the axes but to bias an intrinsic ability of the embryo to initially break symmetry. Furthermore, we suggest that Wnt signalling has a separable activity involved in the elongation of the axis. Highlighted Article: Early gastruloid patterning involves the development of the embryonic axes in the absence of extra-embryonic tissues, a process that relies on the interactions of Nodal and Wnt, but not BMP.

Spatio-Temporal Differences in Dystrophin Dynamics at mRNA and Protein Levels Revealed by a Novel FlipTrap Line

Dystrophin (Dmd) is a structural protein that links the extracellular matrix to actin filaments in muscle fibers and is required for the maintenance of muscles integrity. Mutations in Dmd lead to muscular dystrophies in humans and other vertebrates. Here, we report the characterization of a zebrafish gene trap line that fluorescently labels the endogenous Dmd protein (Dmd-citrine, Gt(dmd-citrine) ct90a). We show that the Dmd-citrine line recapitulates endogenous dmd transcript expression and Dmd protein localization. Using this Dmd-citrine line, we follow Dmd localization to the myosepta in real-time using time-lapse microscopy, and find that the accumulation of Dmd protein at the transverse myosepta coincides with the onset of myotome formation, a critical stage in muscle maturation. We observed that Dmd protein localizes specifically to the myosepta prior to dmd mRNA localization. Additionally, we demonstrate that the Dmd-citrine line can be used to assess muscular dystrophy following both genetic and physical disruptions of the muscle.

Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

ABSTRACT For decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting and/or homogenization. Here, we eliminate the trade-off between quantitation and anatomical context, using quantitative in situ hybridization chain reaction (qHCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable bi-directional queries that open a new era for in situ hybridization. Summary: Quantitative in situ hybridization chain reaction (qHCR) enables quantitative multidimensional analyses of developmental gene expression with subcellular resolution in an anatomical context.