In the trachea and bronchi of the mouse, airway smooth muscle (SM) and cartilage are localized to complementary domains surrounding the airway epithelium. Proper juxtaposition of these tissues ensures a balance of elasticity and rigidity that is critical for effective air passage. It is unknown how this tissue complementation is established during development. Here we dissect the developmental relationship between these tissues by genetically disrupting SM formation (through Srf inactivation) or cartilage formation (through Sox9 inactivation) and assessing the impact on the remaining lineage. We found that, in the trachea and main bronchi, loss of SM or cartilage resulted in an increase in cell number of the remaining lineage, namely the cartilage or SM, respectively. However, only in the main bronchi, but not in the trachea, did the loss of SM or cartilage lead to a circumferential expansion of the remaining cartilage or SM domain, respectively. In addition to SM defects, cartilage-deficient tracheas displayed epithelial phenotypes, including decreased basal cell number, precocious club cell differentiation, and increased secretoglobin expression. These findings together delineate the mechanisms through which a cell-autonomous disruption of one structural tissue can have widespread consequences on upper airway function.airway development | tracheomalacia T he trachea and the main bronchi, here collectively termed the upper airways, are essential conduits of air before and after gas exchange (1, 2). Normal upper airways offer the correct balance of elasticity and rigidity, which together maintain air pressure and prevent airway collapse. The elasticity is primarily provided by smooth muscle (SM) and the rigidity by cartilage. The balance is achieved via a precise juxtaposition of these tissues that together encircle and support the endoderm-derived upper airway epithelium. Clinical evidence suggests that malformation of the SM or cartilage leads to respiratory deficiencies. For example, patients with tracheomalacia and bronchomalacia exhibit airway collapse upon exhalation as a result of softened and disorganized cartilage (3). This collapse causes breathing difficulties that contribute to sleep apnea, respiratory infections, and possibly sudden infant death (4). Despite the importance of a proper SM and cartilage balance, how it is achieved during development is not understood.In mouse upper airways, SM is localized to the dorsal side of the trachea and the medial (inner) sides of the main bronchi. The lower conducting airways lack cartilage and instead SM surrounds the circumference of the epithelial tube. Data suggest that airway SM develops from fibroblast growth factor 10 (Fgf10)-expressing cells found in the distal mesenchyme (5). As the epithelial buds elongate, the Fgf10-expressing cells are thought to translocate proximally along the airway and begin to express SM-specific genes, including SM α-actin-2 (Acta2, also known as SM actin), one of the earliest markers of SM. Additionally, a recent study has u...

Lung development follows a stereotypic program orchestrated by key interactions among epithelial and mesenchymal tissues. Deviations from this developmental program can lead to pulmonary diseases including bronchopulmonary dysplasia and pulmonary hypertension. Significant efforts have been made to examine the cellular and molecular basis of the tissue interactions underlying these stereotypic developmental processes. Genetically engineered mouse models, lung organ culture, and advanced imaging techniques are a few of the tools that have expanded our understanding of the tissue interactions that drive lung development. Intimate crosstalk has been identified between the epithelium and mesenchyme, distinct mesenchymal tissues, and individual epithelial cells types. For interactions such as the epithelial-mesenchymal crosstalk regulating lung specification and branching morphogenesis, the key molecular players, FGF, BMP, WNT, and SHH, are well established. Additionally, VEGF regulation underlies the epithelial-endothelial crosstalk that coordinates airway branching with angiogenesis. Recent work also discovered a novel role for SHH in the epithelial-to-mesenchymal (EMT) transition of the mesothelium. In contrast, the molecular basis for the crosstalk between upper airway cartilage and smooth muscle is not yet known. In this review we examine current evidence of the tissue interactions and molecular crosstalk that underlie the stereotypic patterning of the developing lung and mediate injury repair.

Highlights d Inactivating Myocd prevented airway smooth muscle differentiation and peristalsis d Preventing airway smooth muscle differentiation did not alter epithelial branching d Preventing airway smooth muscle differentiation disrupted tracheal architecture d Preventing airway smooth muscle differentiation led to reduced airway diameter

Little information is available regarding the effects of vitamin D and its metabolites on reproduction in swine. To investigate the effects of feeding the circulating metabolite of vitamin D, 25-hydroxycholecalciferol (25OHD3, ROVIMIX Hy • D, DSM Nutritional Products, Basel, Switzerland) on maternal and fetal circulating 25OHD3 concentration and gilt reproductive performance, a total of 40 PIC Camborough-22 gilts (BW on d -6 = 138 kg) in 4 replicates were randomly assigned to 1 of 2 corn-soybean meal-based diets. The control diet (CTL) was formulated to contain 2,500 IU D3/kg diet, and the experimental diet (25OHD3) was formulated to contain 500 IU D3/kg diet + 50 μg 25OHD3/kg diet. Gilts were fed 2.7 kg of their assigned diet once daily beginning 43 d before breeding. Gilt BW were measured on gestational d -6 and d 90. Gilts were artificially inseminated with PIC 337-G semen 12 h and 24 h after showing signs of estrus. Blood samples were collected from the jugular vein on gestational d -43, -13, 46, and 89 for analysis of circulating 25OHD3 plasma concentration and overall vitamin D status of the gilts. At gestational d 90 ± 1, gilts were harvested and reproductive tracts were removed. Fetal weight, sex, crown-to-rump length (CRL), as well as the number of mummified fetuses were recorded. As expected, circulating plasma concentrations of 25OHD3 were not different among treatment groups at d -43 (CTL = 53.8 ng/mL, 25OHD3 = 57.4 ng/mL; P = 0.66). However, gilts fed 25OHD3 had greater (P < 0.001) circulating plasma concentrations of 25OHD3 on d -13 (89.7 vs. 56.7 ng/mL), d 46 (95.8 vs. 55.7 ng/mL), and d 89 (92.8 vs. 58.2 ng/mL) of gestation compared with CTL-fed gilts. Circulating 25OHD3 was also greater in fetuses from 25OHD3-fed gilts on d 90 (P < 0.001). A 23% increase in pregnancy rate was observed in 25OHD3-fed gilts compared with CTL (78% vs. 55%, respectively; P = 0.21). Maternal BW gain (without conceptus), number of mummified fetuses, mean fetal weight, and mean fetal CRL were similar among treatments (P > 0.05). However, litter size was larger (CTL = 10.2; 25OHD3 = 12.7; P = 0.04) in 25OHD3-fed gilts compared with CTL-fed gilts. Notably, mean fetal weight was not decreased in 25OHD3-fed gilts as frequently occurs when litter size is increased. Overall, feeding 25OHD3 to first-service gilts before and during gestation improved both maternal and fetal vitamin D status and improved maternal reproductive performance.

There is little information available regarding the influence of maternal vitamin D status on fetal skeletal muscle development. Therefore, we investigated the effect of improved vitamin D status resulting from 25-hydroxycholecalciferol (25OHD3) supplementation of dams on fetal skeletal muscle developmental characteristics and myoblast activity using Camborough 22 gilts (n = 40) randomly assigned to 1 of 2 corn-soybean meal-based diets. The control diet (CTL) contained 2,500 IU cholecalciferol (D3)/kg diet, whereas the experimental diet contained 500 IU D3/kg diet plus 50 µg 25OHD3/kg diet. Gilts were fed 2.7 kg of their assigned diet once daily beginning 43 d before breeding through d 90 of gestation. On gestational d 90 (± 1), fetal LM and semitendinosus muscle samples were collected for analysis of developmental characteristics and myoblast activity, respectively. No treatment difference was observed in fetal LM cross-sectional area (P = 0.25). Fetuses from 25OHD3-supplemented gilts had more LM fibers (P = 0.04) that tended to be smaller in cross-sectional area compared with CTL fetuses (P = 0.11). A numerical increase in the total number of Pax7+ myoblasts was also observed in fetuses from 25OHD3-supplemented gilts (P = 0.12). Myoblasts derived from the muscles of fetuses from 25OHD3-fed dams displayed an extended proliferative phase in culture compared with those from fetuses of dams fed only D3 (P < 0.0001). The combination of additional muscle fibers and Pax7+ myoblasts with prolonged proliferative capacity could enhance the postnatal skeletal muscle growth potential of fetuses from 25OHD3-supplemented gilts. These data highlight the importance of maternal vitamin D status on the development of fetal skeletal muscle.