Patterning the embryonic pulmonary mesenchyme.

Smooth muscle guides the morphogenesis of several epithelia during organogenesis, including the mammalian airways. However, it remains unclear how airway smooth muscle differentiation is spatiotemporally patterned and whether it originates from transcriptionally distinct mesenchymal progenitors. Using single-cell RNA-sequencing of embryonic mouse lungs, we show that the pulmonary mesenchyme contains a continuum of cell identities, but no transcriptionally distinct progenitors. Transcriptional variability correlates with spatially distinct sub-epithelial and sub-mesothelial mesenchymal compartments that are regulated by Wnt signaling. Live-imaging and tension-sensors reveal compartment-specific migratory behaviors and cortical forces and show that sub-epithelial mesenchyme contributes to airway smooth muscle. Reconstructing differentiation trajectories reveals early activation of cytoskeletal and Wnt signaling genes. Consistently, Wnt activation induces the earliest stages of smooth muscle differentiation and local accumulation of mesenchymal F-actin, which influences epithelial morphology. Our single-cell approach uncovers the principles of pulmonary mesenchymal patterning and identifies a morphogenetically active mesenchymal layer that sculpts the airway epithelium.

Transmural pressure signals through retinoic acid to regulate lung branching.

During development, the mammalian lung undergoes several rounds of branching, the rate of which is tuned by the relative pressure of the fluid within the lumen of the lung. We carried out bioinformatics analysis of RNA-sequencing of embryonic mouse lungs cultured under physiologic or sub-physiologic transmural pressure and identified transcription factor-binding motifs near genes whose expression changes in response to pressure. Surprisingly, we found retinoic acid (RA) receptor binding sites significantly overrepresented in the promoters and enhancers of pressure-responsive genes. Consistently, increasing transmural pressure activates RA signaling, and pharmacologically inhibiting RA signaling decreases airway epithelial branching and smooth muscle wrapping. We found that pressure activates RA signaling through the mechanosensor Yap. A computational model predicts that mechanical signaling through Yap and RA affects lung branching by altering the balance between epithelial proliferation and smooth muscle wrapping, which we test experimentally. Our results reveal that transmural pressure signals through RA to balance the relative rates of epithelial growth and smooth muscle differentiation in the developing mouse lung and identify RA as a previously unreported component in the mechanotransduction machinery of embryonic tissues.

Negative Transpulmonary Pressure Disrupts Airway Morphogenesis by Suppressing Fgf10.

Mechanical forces are increasingly recognized as important determinants of cell and tissue phenotype and also appear to play a critical role in organ development. During the fetal stages of lung morphogenesis, the pressure of the fluid within the lumen of the airways is higher than that within the chest cavity, resulting in a positive transpulmonary pressure. Several congenital defects decrease or reverse transpulmonary pressure across the developing airways and are associated with a reduced number of branches and a correspondingly underdeveloped lung that is insufficient for gas exchange after birth. The small size of the early pseudoglandular stage lung and its relative inaccessibility in utero have precluded experimental investigation of the effects of transpulmonary pressure on early branching morphogenesis. Here, we present a simple culture model to explore the effects of negative transpulmonary pressure on development of the embryonic airways. We found that negative transpulmonary pressure decreases branching, and that it does so in part by altering the expression of fibroblast growth factor 10 (Fgf10). The morphogenesis of lungs maintained under negative transpulmonary pressure can be rescued by supplementing the culture medium with exogenous FGF10. These data suggest that Fgf10 expression is regulated by mechanical stress in the developing airways. Understanding the mechanical signaling pathways that connect transpulmonary pressure to FGF10 can lead to the establishment of novel non-surgical approaches for ameliorating congenital lung defects.

Local accumulation of extracellular matrix regulates global morphogenetic patterning in the developing mammary gland.

The tree-like pattern of the mammary epithelium is formed during puberty through a process known as branching morphogenesis. Although mammary epithelial branching is stochastic and generates an epithelial tree with a random pattern of branches, the global orientation of the developing epithelium is predictably biased along the long axis of the gland. Here, we combine analysis of pubertal mouse mammary glands, a three-dimensional (3D)-printed engineered tissue model, and computational models of morphogenesis to investigate the origin and the dynamics of the global bias in epithelial orientation during pubertal mammary development. Confocal microscopy analysis revealed that a global bias emerges in the absence of pre-aligned networks of type I collagen in the fat pad and is maintained throughout pubertal development until the widespread formation of lateral branches. Using branching and annihilating random walk simulations, we found that the angle of bifurcation of terminal end buds (TEBs) dictates both the dynamics and the extent of the global bias in epithelial orientation. Our experimental and computational data demonstrate that a local increase in stiffness from the accumulation of extracellular matrix, which constrains the angle of bifurcation of TEBs, is sufficient to pattern the global orientation of the developing mammary epithelium. These data reveal that local mechanical properties regulate the global pattern of mammary epithelial branching and may provide new insight into the global patterning of other branched epithelia.

Smooth muscle: a stiff sculptor of epithelial shapes.

Smooth muscle is increasingly recognized as a key mechanical sculptor of epithelia during embryonic development. Smooth muscle is a mesenchymal tissue that surrounds the epithelia of organs including the gut, blood vessels, lungs, bladder, ureter, uterus, oviduct and epididymis. Smooth muscle is stiffer than its adjacent epithelium and often serves its morphogenetic function by physically constraining the growth of a proliferating epithelial layer. This constraint leads to mechanical instabilities and epithelial morphogenesis through buckling. Smooth muscle stiffness alone, without smooth muscle cell shortening, seems to be sufficient to drive epithelial morphogenesis. Fully understanding the development of organs that use smooth muscle stiffness as a driver of morphogenesis requires investigating how smooth muscle develops, a key aspect of which is distinguishing smooth muscle-like tissues from one another in vivo and in culture. This necessitates a comprehensive appreciation of the genetic, anatomical and functional markers that are used to distinguish the different subtypes of smooth muscle (for example, vascular versus visceral) from similar cell types (including myofibroblasts and myoepithelial cells). Here, we review how smooth muscle acts as a mechanical driver of morphogenesis and discuss ways of identifying smooth muscle, which is critical for understanding these morphogenetic events.This article is part of the Theo Murphy meeting issue 'Mechanics of Development'.

Microfluidic chest cavities reveal that transmural pressure controls the rate of lung development.

Mechanical forces are increasingly recognized to regulate morphogenesis, but how this is accomplished in the context of the multiple tissue types present within a developing organ remains unclear. Here, we use bioengineered 'microfluidic chest cavities' to precisely control the mechanical environment of the fetal lung. We show that transmural pressure controls airway branching morphogenesis, the frequency of airway smooth muscle contraction, and the rate of developmental maturation of the lungs, as assessed by transcriptional analyses. Time-lapse imaging reveals that branching events are synchronized across distant locations within the lung, and are preceded by long-duration waves of airway smooth muscle contraction. Higher transmural pressure decreases the interval between systemic smooth muscle contractions and increases the rate of morphogenesis of the airway epithelium. These data reveal that the mechanical properties of the microenvironment instruct crosstalk between different tissues to control the development of the embryonic lung.

An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa.

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO2-) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO3-) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO3-→NO2-→NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O2, NO2-, and NO3- in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO3-, which was relieved upon O2 depletion. As cell growth depleted dissolved O2 levels, NO3- was converted to NO2- at near-stoichiometric levels, whereas NO2- consumption did not coincide with NO or NO3- accumulation. Assimilatory NO2- reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.

Optical systems for point-of-care diagnostic instrumentation: analysis of imaging performance and cost.

One of the key elements in point-of-care (POC) diagnostic test instrumentation is the optical system required for signal detection and/or imaging. Many tests which use fluorescence, absorbance, or colorimetric optical signals are under development for management of infectious diseases in resource limited settings, where the overall size and cost of the device is of critical importance. At present, high-performance lenses are expensive to fabricate and difficult to obtain commercially, presenting barriers for developers of in vitro POC tests or microscopic image-based diagnostics. We recently described a compact "hybrid" objective lens incorporating both glass and plastic optical elements, with a numerical aperture of 1.0 and field-of-view of 250 µm. This design concept may potentially enable mass-production of high-performance, low-cost optical systems which can be easily incorporated in the readout path of existing and emerging POC diagnostic assays. In this paper, we evaluate the biological imaging performance of these lens systems in three broad POC diagnostic application areas; (1) bright field microscopy of histopathology slides, (2) cytologic examination of blood smears, and (3) immunofluorescence imaging. We also break down the fabrication costs and draw comparisons with other miniature optical systems. The hybrid lenses provided images with quality comparable to conventional microscopy, enabling examination of neoplastic pathology and infectious parasites including malaria and cryptosporidium. We describe how these components can be produced at below $10 per unit in full-scale production quantities, making these systems well suited for use within POC diagnostic instrumentation.