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Moffitt Lab @moffittlab.bsky.social · Jul 31

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

Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · Jul 31

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 · Jul 31

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/

Moffitt Lab @moffittlab.bsky.social · Jul 31

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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Moffitt Lab @moffittlab.bsky.social · May 30

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!

Moffitt Lab @moffittlab.bsky.social · May 28

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

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Moffitt Lab @moffittlab.bsky.social · May 28

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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Moffitt Lab @moffittlab.bsky.social · May 28

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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Moffitt Lab @moffittlab.bsky.social · May 28

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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Moffitt Lab @moffittlab.bsky.social · May 28

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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Moffitt Lab @moffittlab.bsky.social · May 28

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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Moffitt Lab @moffittlab.bsky.social · May 28

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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Moffitt Lab @moffittlab.bsky.social · May 28

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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Moffitt Lab @moffittlab.bsky.social · May 28

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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Moffitt Lab @moffittlab.bsky.social · May 28

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