Inna Averbukh

Abstract Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR) 1–3 . However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal–fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells.

The intestinal epithelium is a structured organ composed of crypts harboring Lgr5+ stem cells, and villi harboring differentiated cells. Spatial transcriptomics have demonstrated profound zonation of epithelial gene expression along the villus axis, but the mechanisms shaping this spatial variability are unknown. Here, we combine laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These include villus tip telocytes (VTTs) that express Lgr5, a gene previously considered a specific crypt epithelial stem cell marker. VTTs are elongated cells that line the villus tip epithelium and signal through Bmp morphogens and the non-canonical Wnt5a ligand. Their ablation is associated with perturbed zonation of enterocyte genes induced at the villus tip. Our study provides a spatially-resolved cell atlas of the small intestinal stroma and exposes Lgr5+ villus tip telocytes as regulators of the epithelial spatial expression programs along the villus axis.

Morphogen gradients guide the patterning of tissues and organs during the development of multicellular organisms. In many cases, morphogen signaling is also required for tissue growth. The consequences of this interplay between growth and patterning are not well understood. In the Drosophila wing imaginal disc, the morphogen Dpp guides patterning and is also required for tissue growth. In particular, it was recently reported that cell division in the disc correlates with the temporal increase in Dpp signaling. Here we mathematically model morphogen gradient formation in a growing tissue, accounting also for morphogen advection and dilution. Our analysis defines a new scaling mechanism, which we term the morphogen-dependent division rule (MDDR): when cell division depends on the temporal increase in morphogen signaling, the morphogen gradient scales with the growing tissue size, tissue growth becomes spatially uniform and the tissue naturally attains a finite size. This model is consistent with many properties of the wing disc. However, we find that the MDDR is not consistent with the phenotype of scaling-defective mutants, supporting the view that temporal increase in Dpp signaling is not the driver of cell division during late phases of disc development. More generally, our results show that local coupling of cell division with morphogen signaling can lead to gradient scaling and uniform growth even in the absence of global feedbacks. The MDDR scaling mechanism might be particularly beneficial during rapid proliferation, when global feedbacks are hard to implement.

Single-cell RNA sequencing combined with spatial information on landmark genes enables reconstruction of spatially-resolved tissue cell atlases. However, such approaches are challenging for rare cell types, since their mRNA contents are diluted in the spatial transcriptomics bulk measurements used for landmark gene detection. In the small intestine, enterocytes, the most common cell type, exhibit zonated expression programs along the crypt-villus axis, but zonation patterns of rare cell types such as goblet and tuft cells remain uncharacterized. Here, we present ClumpSeq, an approach for sequencing small clumps of attached cells. By inferring the crypt-villus location of each clump from enterocyte landmark genes, we establish spatial atlases for all epithelial cell types in the small intestine. We identify elevated expression of immune-modulatory genes in villus tip goblet and tuft cells and heterogeneous migration patterns of enteroendocrine cells. ClumpSeq can be applied for reconstructing spatial atlases of rare cell types in other tissues and tumors.