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Taken together, these results underscore the potential of our new murine gut atlas to inform a broad range of biological and biomedical questions! 12/12

Remarkably, many aspects of the gut were largely unchanged! Nonetheless, we observed profound shifts in immune cell abundance and distribution—highlighting the central role of the microbiota in shaping and educating the immune system. 11/

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Finally, while the microbiota shapes nearly every aspect of host physiology, its impact on the molecular, cellular, and spatial structure of the gut remained unclear. To address this, we rebuilt our entire atlas using mice lacking a microbiota. 10/

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The gut microbiota produces metabolites that impact host immunity and even CNS function—but how? Using a near-comprehensive receptor panel, we mapped metabolite sensitivity across gut cell types, revealing rich hypotheses for cell mediated microbiota–host interactions! 9/

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We also found a surprising set of genes with spatially patchy expression! In a great collaboration with Phillip Nicol (Irizarry lab), we developed a method to detect these patches —revealing insights into interferon signaling and epithelial specialization near GALT! 8/

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This mucosal ruler revealed that nearly all cell populations fine-tune their gene expression based on their micron-scale position in the mucosa. Importantly, once one knows what to look for, these spatial signatures are also detectable in single-cell RNA-seq data! 7/

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Another surprise was the intricate spatial organization of cells and RNAs in the mucosa. From crypt base to lumen, we observed gradients in cell types and gene expression, some known, many novel! We created an expression-based mucosal spatial ruler to capture this pattern. 6/

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Our atlas revealed multiple surprises! For instance, we found a novel mature enterocyte marked by Scnn1g, a subunit of the epithelial sodium channel, and the pseudogene Best4-ps —suggesting it might be the long-sought mouse homolog of the human BEST4+ enterocyte! 5/

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Excitingly, these advances enabled us to define a remarkable diversity of cell populations—78 in total—with finely resolved subdivisions within immune cells, fibroblasts, and the enteric nervous system! 4/

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As small molecule receptors are often lowly expressed even when functional, we developed new approaches to improve the sensitivity of our MERFISH measurements, including an improved cell segmentation pipeline. 3/

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The mammalian gut is home to a massive diversity of microbe-, diet-, and host-derived small molecules. To provide new insights into the cellular and spatial organization of sensation, we profiled 2.1M cells across four gut regions and two microbiome states! 2/

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Excited to share our new work from Rosalind Xu, where we used MERFISH to build a single-cell spatial transcriptomic atlas of the mouse gut—and to explore host-microbiome interactions in situ! www.biorxiv.org/content/10.1... 1/

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Moffitt Lab @moffittlab.bsky.social · 30/05/2025

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Yesterday our first grad student -- DR. Rosalind Xu -- had her official PhD hooding ceremony in Harvard CCB! A massive congratulations to her and all of the other freshly minted PhDs!

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As plasma cells play fundamental roles in tissue behavior in health and disease, we are excited to see the wide range of questions that BCR-MERFISH will now allow us and others to address! 9/9

To our surprise, we found that some plasma cell clones form patches of co-occurring clones along the gut, suggesting, perhaps, that there is local production of IgA specific to a subset of the microbiome! 8/9

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Plasma cells modulate the microbiome by secreting IgA, and different plasma cell clones secrete IgA that targets different bacteria. But how these cells are distributed along the gut is not clear. Excitingly, BCR-MERFISH can now measure this distribution. 7/9

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Importantly, BCR-MERFISH labels not only the Clone ID, but also the transcriptome of all surrounding cell types in the same slice, placing unique clones within their tissue contexts with single-cell resolution. 6/9

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To test BCR-MERFISH, we profiled V gene usage in plasma B cells in the mouse ileum, defined B cell clones via the VH and VKVL gene choice within individual B cells, and showed that the usage of these genes agreed with BCR-sequencing. 5/9

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However, MERFISH has been unable to discriminate B cells based on the BCR sequence because of the high degree of homology between V, D, and J genes. Evan solved this problem with a homology-aware probe design and encoding approach to produce a technique we call BCR-MERFISH! 4/9

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However, some cell types - such as plasma B cells – may only differ in their expression of the two transcripts that form the B cell receptor (BCR). Yet, it is the sequence of this receptor, generated through V(D)J recombination, that sets their antigen specificity! 3/9

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Multiplexed FISH methods such as #MERFISH can image 1000s of unique RNA transcripts within single cells, and in turn define a massive diversity of cell types and states within intact tissues. 2/9

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Proud to announce our new technique for tracking relative B cell clonality in situ with B Cell Receptor MERFISH (BCR-MERFISH)! Congratulations to Evan Yang and our colleagues in the Carroll laboratory! Check out our bioRxiv: www.biorxiv.org/content/10.1... 1/9

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However, some cell types - such as plasma B cells – may only differ in their expression of two transcripts that form the B cell receptor (BCR). Yet, it is the sequence of this receptor, generated through V(D)J recombination, that sets their antigen specificity! 3/9

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Moffitt Lab @moffittlab.bsky.social · 23/03/2025

Multiplexed FISH methods such as #MERFISH can image 1000s of unique RNA transcripts within single cells, and in turn define a massive diversity of cell types and states within intact tissues. 2/9

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Thrilled to share our newest work! A very talented research scientist in our lab, Josh Luce, as been re-examining protocol choices for MERFISH. Through his hard work we now have a revised protocol set that improves performance for long measurements. Read more here: www.biorxiv.org/content/10.1...

biorxiv.org

Protocol Optimization Improves the Performance of Multiplexed RNA Imaging

Spatial transcriptomics has emerged as a powerful tool to define the cellular structure of diverse tissues. One such method is multiplexed error robust fluorescence in situ hybridization (MERFISH). ME...

Moffitt Lab @moffittlab.bsky.social · 18/03/2025

There is still time to register for this keystone meeting! The talk line-up is amazing, and it will only get better with speakers selected from the abstracts! youtu.be/H4t4tML35Ic

youtu.be

Single Cell Biology Meeting Promo

YouTube video by KeystoneSymposia

Moffitt Lab @moffittlab.bsky.social · 07/02/2025

Interested in connecting single-cell biology to tissue level behaviors? I have the Keystone meeting for you! Short-talk abstracts are due on Feb 11th. Registration continues to April 17th! www.keystonesymposia.org/conferences/...

keystonesymposia.org

Single Cell Biology: Unique Cells to Tissue Ecosystems | Keystone Symposia

Join us at the Keystone Symposia on Single Cell Biology: Unique Cells to Tissue Ecosystems, May 2025, in Whistler, with field leaders!

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Whether it is specialization in biofilms, the dynamics of antibiotic-resistant persisters, or complex communication in commensal communities, we anticipate bacterial-MERFISH will be a powerful tool for a wide range of questions! Excited to see this technique out! 13/

Remarkably, B. theta adapts its gene expression to different niches in the colon. For example, when near the host mucus layer, B. theta upregulates a rich diversity of genes associated with the harvest of mucus polysaccharides (PULs)! 12/

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Most bacteria live in complex, spatially structured environments not well approximated by a culture flask. To illustrate the ability of bacterial-MERFISH to profile bacterial gene expression in such environments, we characterized B. theta expression in the colon of mice. 11/

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The results were a shock! The E. coli transcriptome is extensively organized. We identified and classified a diversity of patterns and found that elements of the proteome organization and the genome organization shape the organization of the transcriptome. 10/

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Even though bacteria are small, there is a growing appreciation that different mRNAs can be localized to different sub-cellular regions with important functional consequences. So next we charted the organization of the E. coli transcriptome with bacterial-MERFISH. 9/

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This heterogeneous response taught us two things. First, the required levels of essential genes during growth arises from coordinated transcriptional bursts. Second, when starved, E. coli explores many different sugars--not in our medium--before settling on xylose. 8/

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We next revisited a classic experiment. E. coli grown in glucose and xylose will first consume glucose, then pause growth, before growing again on xylose. Classically, this pause is the time to make xylose-associated genes, but we observed a richer single-cell response. 7/

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Expanded bacteria are then combined with #MERFISH to image large fractions of the transcriptome. Benchmarked in E. coli, bacterial-MERFISH is accurate across a large dynamic range, has high detection efficiency and low false positives, and can image 100k+ cells. 6/

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However, three orders of magnitude didn’t seem so daunting to Ari, Yuanyou, and Nana, so they built a bacteria-optimized #ExpansionMicroscopy toolbox that is capable of tunable expansion of individual bacteria up to 1000-fold in volume! 5/

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However, until now, these methods have been incompatible with an entire domain of life—bacteria! The reason is that multiplexed imaging requires resolving single molecules, and bacterial mRNAs are just way too dense--three orders of magnitude too dense! 4/

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Image-based approaches to spatial transcriptomics — like #MERFISH — allow the massively multiplexed imaging of individual mRNAs within single cells within intact tissue samples. As such they have offered many exciting insights into a wide variety of eukaryotic questions! 3/

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