Wan Kee Chock

Spatial omics data are clustered to define both cell types and tissue domains. We present Building Aggregates with a Neighborhood Kernel and Spatial Yardstick (BANKSY), an algorithm that unifies these two spatial clustering problems by embedding cells in a product space of their own and the local neighborhood transcriptome, representing cell state and microenvironment, respectively. BANKSY's spatial feature augmentation strategy improved performance on both tasks when tested on diverse RNA (imaging, sequencing) and protein (imaging) datasets. BANKSY revealed unexpected niche-dependent cell states in the mouse brain and outperformed competing methods on domain segmentation and cell typing benchmarks. BANKSY can also be used for quality control of spatial transcriptomics data and for spatially aware batch effect correction. Importantly, it is substantially faster and more scalable than existing methods, enabling the processing of millions of cell datasets. In summary, BANKSY provides an accurate, biologically motivated, scalable and versatile framework for analyzing spatially resolved omics data.

Abstract Each cell type in a solid tissue has a characteristic transcriptome and spatial arrangement, both of which are observable using modern spatial omics assays. However, the common practice is still to ignore spatial information when clustering cells to identify cell types. In fact, spatial location is typically considered only when solving the related, but distinct, problem of demarcating tissue domains (which could include multiple cell types). We present BANKSY, an algorithm that unifies cell type clustering and domain segmentation by constructing a product space of cell and neighbourhood transcriptomes, representing cell state and microenvironment, respectively. BANKSY’s spatial kernel-based feature augmentation strategy improves per-formance and scalability on both tasks when tested on FISH-based and sequencing-based spatial omics data. Uniquely, BANKSY identified hitherto undetected niche-dependent cell states in two mouse brain regions. Lastly, we show that quality control of spatial omics data can be formulated as a domain identification problem and solved using BANKSY. BANKSY represents a biologically motivated, scalable, and versatile framework for analyzing spatial omics data.

BACKGROUND: Cardiomyocyte differentiation involves a stepwise clearance of repressors and fate-restricting regulators through the modulation of BMP (bone morphogenic protein)/Wnt-signaling pathways. However, the mechanisms and how regulatory roadblocks are removed with specific developmental signaling pathways remain unclear. METHODS: We conducted a genome-wide CRISPR screen to uncover essential regulators of cardiomyocyte specification in human embryonic stem cells using a myosin heavy chain 6 ( MYH6 )-GFP (green fluorescence protein) reporter system. After an independent secondary single guide ribonucleic acid validation of 25 candidates, we identified NF2 (neurofibromin 2), a moesin-ezrin-radixin like (MERLIN) tumor suppressor, as an upstream driver of early cardiomyocyte lineage specification. Independent monoclonal NF2 knockouts were generated using CRISPR-Cas9, and cell states were inferred through bulk RNA sequencing and protein expression analysis across differentiation time points. Terminal lineage differentiation was assessed by using an in vitro 2-dimensional-micropatterned gastruloid model, trilineage differentiation, and cardiomyocyte differentiation. Protein interaction and post-translation modification of NF2 with its interacting partners were assessed using site-directed mutagenesis, coimmunoprecipitation, and proximity ligation assays. RESULTS: Transcriptional regulation and trajectory inference from NF2 -null cells reveal the loss of cardiomyocyte identity and the acquisition of nonmesodermal identity. Sustained elevation of early mesoderm lineage repressor SOX2 and upregulation of late anticardiac regulators CDX2 and MSX1 in NF2 knockout cells reflect a necessary role for NF2 in removing regulatory roadblocks. Furthermore, we found that NF2 and AMOT (angiomotin) cooperatively bind to YAP (yes-associated protein) during mesendoderm formation, thereby preventing YAP activation, independent of canonical MST (mammalian sterile 20-like serine-threonine protein kinase)–LATS (large tumor suppressor serine-threonine protein kinase) signaling. Mechanistically, cardiomyocyte lineage identity was rescued by wild-type and NF2 serine-518 phosphomutants, but not NF2 FERM (ezrin-radixin-meosin homology protein) domain blue-box mutants, demonstrating that the critical FERM domain–dependent formation of the AMOT-NF2-YAP scaffold complex at the adherens junction is required for early cardiomyocyte lineage differentiation. CONCLUSIONS: These results provide mechanistic insight into the essential role of NF2 during early epithelial-mesenchymal transition by sequestering the repressive effect of YAP and relieving regulatory roadblocks en route to cardiomyocytes.