Probes for INS

Positional information encoded in signaling molecules is essential for early patterning in the prosensory domain of the developing cochlea. The sensory epithelium, the organ of Corti, contains an exquisite repeating pattern of hair cells and supporting cells. This requires precision in the morphogen signals that set the initial radial compartment boundaries, but this has not been investigated. To measure gradient formation and morphogenetic precision in developing cochlea, we developed a quantitative image analysis procedure measuring SOX2 and pSMAD1/5/9 profiles in mouse embryos at embryonic day (E)12.5, E13.5, and E14.5. Intriguingly, we found that the pSMAD1/5/9 profile forms a linear gradient up to the medial ~ 75% of the PSD from the pSMAD1/5/9 peak in the lateral edge during E12.5 and E13.5. This is a surprising activity readout for a diffusive BMP4 ligand secreted from a tightly constrained lateral region since morphogens typically form exponential or power-law gradient shapes. This is meaningful for gradient interpretation because while linear profiles offer the theoretically highest information content and distributed precision for patterning, a linear morphogen gradient has not yet been observed. Furthermore, this is unique to the cochlear epithelium as the pSMAD1/5/9 gradient is exponential in the surrounding mesenchyme. In addition to the information-optimized linear profile, we found that while pSMAD1/5/9 is stable during this timeframe, an accompanying gradient of SOX2 shifts dynamically. Last, through joint decoding maps of pSMAD1/5/9 and SOX2, we see that there is a high-fidelity mapping between signaling activity and position in the regions that will become Kölliker's organ and the organ of Corti. Mapping is ambiguous in the prosensory domain precursory to the outer sulcus. Altogether, this research provides new insights into the precision of early morphogenetic patterning cues in the radial cochlea prosensory domain.

Background & Aims The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organised inside glands and crypts. Regeneration depends on WNT/β-Catenin pathway activation, but to understand homeostasis and its dysregulation in disease we need to identify the signalling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen- and gastric acid- producing cells. Methods We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief and parietal cells. We localised the source of the growth factors in the tissue of origin. Results We show that EGF is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with BMP/NOGGIN signals, EGF controls the differentiation of foveolar cells vs. parietal or chief cells. We also show that EGF is likely to underlie alteration of the gastric mucosa in the pre-cancerous condition atrophic gastritis. Conclusions Use of our recently established mucosoid cultures in combination with analysis of the tissue-of-origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signalling microenvironment in the human gastric glands.

Branching morphogenesis is a key process essential for lung and other organ development in which cellular and tissue architecture branch out to maximize surface area. While this process is known to be regulated by differential gene expression of ligands and receptors, how chromatin remodeling regulates this process remains unclear. Znhit1, acting as a chromatin remodeler, has previously been shown to control the deposition of the histone variant H2A.Z. Here, we demonstrate that Znhit1 also plays an important role in regulating lung branching. Using Znhit1 conditional knockout mice, we show that Znhit1 deficiency in the embryonic lung epithelium leads to failure of branching morphogenesis and neonatal lethality, which is accompanied by reduced cell proliferation and increased cell apoptosis of the epithelium. The results from the transcriptome and the ChIP assay reveal that this is partially regulated by the derepression of Bmp4, encoding bone morphogenetic protein 4, which is a direct target of H2A.Z. Furthermore, we show that inhibition of BMP signaling by the protein inhibitor Noggin rescues the lung branching defects of Znhit1 mutants ex vivo. Taken together, our study identifies the critical role of Znhit1/H2A.Z in embryonic lung morphogenesis via the regulation of BMP signaling.

The lung alveolus is lined with alveolar type 1 (AT1) and type 2 (AT2) epithelial cells. During alveologenesis, increasing demand associated with expanding alveolar numbers is met by proliferating progenitor AT2s (pAT2). Little information exists regarding the identity of this population and their niche microenvironment. We show that during alveologenesis, Hedgehog-responsive PDGFRa(+) progenitors (also known as SCMFs) are a source of secreted trophic molecules that maintain a unique pAT2 population. SCMFs are in turn maintained by TGFβ signaling. Compound inactivation of Alk5 TβR2 in SCMFs reduced their numbers and depleted the pAT2 pool without impacting differentiation of daughter cells. In lungs of preterm infants who died with bronchopulmonary dysplasia, PDGFRa is reduced and the number of proliferative AT2s is diminished, indicating that an evolutionarily conserved mechanism governs pAT2 behavior during alveologenesis. SCMFs are a transient cell population, active only during alveologenesis, making them a unique stage-specific niche mesodermal cell type in mammalian organs.

It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.