SUMMARY Ductal carcinoma in situ (DCIS) is a pre-invasive lesion that is thought to be a precursor to invasive breast cancer (IBC). To understand the changes in the tumor microenvironment (TME) accompanying transition to IBC, we used multiplexed ion beam imaging by time of flight (MIBI-TOF) and a 37-plex antibody staining panel to interrogate 79 clinically annotated surgical resections using machine learning tools for cell segmentation, pixel-based clustering, and object morphometrics. Comparison of normal breast with patient-matched DCIS and IBC revealed coordinated transitions between four TME states that were delineated based on the location and function of myoepithelium, fibroblasts, and immune cells. Surprisingly, myoepithelial disruption was more advanced in DCIS patients that did not develop IBC, suggesting this process could be protective against recurrence. Taken together, this HTAN Breast PreCancer Atlas study offers insight into drivers of IBC relapse and emphasizes the importance of the TME in regulating these processes.

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.

Patterning by morphogen gradients relies on the capacity to generate reproducible distribution profiles. Morphogen spread depends on kinetic parameters, including diffusion and degradation rates, which vary between embryos, raising the question of how variability is controlled. We examined this in the context of Toll-dependent dorsoventral (DV) patterning of the Drosophila embryo. We find that low embryo-to-embryo variability in DV patterning relies on wntD, a Toll-target gene expressed initially at the posterior pole. WntD protein is secreted and disperses in the extracellular milieu, associates with its receptor Frizzled4, and inhibits the Toll pathway by blocking the Toll extracellular domain. Mathematical modeling predicts that WntD accumulates until the Toll gradient narrows to its desired spread, and we support this feedback experimentally. This circuit exemplifies a broadly applicable induction-contraction mechanism, which reduces patterning variability through a restricted morphogen-dependent expression of a secreted diffusible inhibitor.

15Biological timers synchronize patterning processes during embryonic development. In the

AbstractThe 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 combined laser capture micro-dissection and single cell RNA sequencing to uncover spatially zonated populations of mesenchymal cells along the crypt-villus axis. These included 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 strongly perturbs the 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.

Biological timers synchronize patterning processes during embryonic development. In the Drosophila embryo, neural progenitors (neuroblasts; NBs) produce a sequence of unique neurons whose identities depend on the sequential expression of temporal transcription factors (TTFs). The stereotypy and precision of NB lineages indicate reproducible TTF timer progression. We combine theory and experiments to define the timer mechanism. The TTF timer is commonly described as a relay of activators, but its regulatory circuit is also consistent with a repressor-decay timer, where TTF expression begins when its repressor decays. Theory shows that repressor-decay timers are more robust to parameter variations than activator-relay timers. This motivated us to experimentally compare the relative importance of the relay and decay interactions in vivo. Comparing WT and mutant NBs at high temporal resolution, we show that the TTF sequence progresses primarily by repressor-decay. We suggest that need for robust performance shapes the evolutionary-selected designs of biological circuits.

Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible extracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, we followed the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. We conclude that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos.