Various factors play an essential role in patterning the digestive tract. During development, Sox2 and Cdx2 are exclusively expressed in the anterior and the posterior parts of the primitive gut, respectively. However, it is unclear whether these transcription factors influence each other in determining specification of the naïve gut endoderm. We therefore investigated whether Sox2 redirects the fate of the prospective intestinal part of the primitive gut. Ectopic expression of Sox2 in the posterior region of the primitive gut caused anteriorization of the gut toward a gastric-like phenotype. Sox2 activated the foregut transcriptional program, in spite of sustained co-expression of endogenous Cdx2. However, binding of Cdx2 to its genomic targets and thus its transcriptional activity was strongly reduced. Recent findings indicate that endodermal Cdx2 is required to initiate the intestinal program and to suppress anterior cell fate. Our findings suggest that reduced Cdx2 expression by itself is not sufficient to cause anteriorization, but that Sox2 expression is also required. Moreover, it indicates that the balance between Sox2 and Cdx2 function is essential for proper specification of the primitive gut and that Sox2 may overrule the initial patterning of the primitive gut, emphasizing the plasticity of the primitive gut.
Lung development is determined by the coordinated expression of several key genes. Previously, we and others have shown the importance of the sex determining region Y-box 2 (Sox2) gene in lung development. Transgenic expression of Sox2 during lung development resulted in cystic airways, and here we show that modulating the timing of ectopic Sox2 expression in the branching regions of the developing lung results in variable cystic lesions resembling the spectrum of the human congenital disorder congenital cystic adenomatoid malformation (CCAM). Sox2 dominantly differentiated naive epithelial cells into the proximal lineage irrespective of the presence of Fgf10. Sox2 directly induced the expression of Trp63, the master switch toward the basal cell lineage and induced the expression of Gata6, a factor involved in the emergence of bronchoalveolar stem cells. We showed that SOX2 and TRP63 are coexpressed in the lungs of human patients with type II CCAM. The combination of premature differentiation toward the proximal cell lineage and the induction of proliferation resulted in the cyst-like structures. Thus, we show that Sox2 is directly responsible for the emergence of two lung progenitor cells: basal cells by regulating the master gene Trp63 and bronchoalveolar stem cells by regulating Gata6.
Alveolarization of the developing lung is an important step toward the switch from intrauterine life to breathing oxygen-rich air after birth. The distal airways structurally change to minimize the gas exchange path, and Type II pneumocytes increase the production of surfactants, which are required to reduce surface tension at the air-liquid interface in the alveolus. Hypoxia-inducible factor 2α (Hif2α) is an oxygen-regulated transcription factor expressed in endothelial and Type II cells, and its expression increases toward the end of gestation. We investigated the role of Hif2α in Type II cells by conditionally expressing an oxygen-insensitive mutant of Hif2α in airway epithelial cells during development. Newborn mice expressing the mutant Hif2α were born alive but quickly succumbed to respiratory distress. Subsequent analysis of the lungs revealed dilated alveoli covered with enlarged, aberrant Type II cells and a diminished number of Type I cells. The Type II cells accumulated glycogen in part by increased glucose uptake via the up-regulation of the glucose transporter 1. Furthermore, the cells lacked two crucial enzymes involved in the metabolism of glycogen into surfactant lipids, lysophosphatidylcholine acyltransferase and ATP-binding cassette sub-family A member 3. We conclude that Hif2α is a key regulator in alveolar maturation and the production of phospholipids by Type II cells.
Patients with congenital diaphragmatic hernia (CDH) often suffer from severe pulmonary hypertension, and the choice of current vasodilator therapy is mostly based on trial and error. Because pulmonary vascular abnormalities are already present early during development, we performed a study to modulate these pulmonary vascular changes at an early stage during gestation. Pregnant Sprague-Dawley rats were treated with nitrofen at day 9.5 of gestation (E9.5) to induce CDH in the offspring, and subsequently, the phosphodiesterase-5 inhibitor sildenafil and/or the novel prostaglandin-I receptor agonist selexipag (active compound NS-304) were administered from E17.5 until E20.5. The clinical relevant start of the treatment corresponds to week 20 of gestation in humans, when CDH is usually detected by ultrasound. CDH pups showed increased density of air saccules that was reverted after the use of only sildenafil. The pulmonary vascular wall was thickened, and right ventricular hypertrophy was present in the CDH group and improved both after single treatment with sildenafil or selexipag, whereas the combination therapy with both compounds did not have additive value. In conclusion, antenatal treatment with sildenafil improved airway morphogenesis and pulmonary vascular development, whereas selexipag only acted positively on pulmonary vascular development. The combination of both compounds did not act synergistically, probably because of a decreased efficiency of both compounds caused by cytochrome- P450 3A4 interaction and induction. These new insights create important possibilities for future treatment of pulmonary vascular abnormalities in CDH patients already in the antenatal period of life.
SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium.
The mortality and morbidity of patients with congenital diaphragmatic hernia (CDH) is primarily caused by treatment-resistant, persistent pulmonary hypertension. Structural vascular changes, exemplified by extensive muscularization, are already present early in gestation, but the origin of these abnormalities is unknown. Understanding the origin of the vascular defects is important to improve treatment modalities. Here, we show that the distribution of pericytes is different and may thereby potentially initiate the vascular pathology in CDH. Transient inhibition of retinoic acid (RA) signaling early during pregnancy, the basis of the CDH mouse model, led to an increase in the number of pericytes, thereby affecting the angiogenic potential of pericytes in the fetuses. Pericytes of CDH lungs showed reduced proliferation and an increased ACTA2 expression, which indicates that these pericytes are more contractile than in control lung pericytes. This resulted in increased pericyte coverage of pulmonary vessels and reduced expansion of the capillary bed, the earliest pathological sign of the structural changes in CDH. Furthermore, the pericytes had reduced and altered collagen IV deposition in CDH, pointing to a loss of basal membrane integrity between pericytes and endothelial cells. Inhibition of RA signaling in vitro resulted in reduced migration of pericytes, reduced angiogenesis, and loss of collagen IV expression. Importantly, we confirmed our findings in lungs of human CDH patient samples. In summary, inhibition of RA signaling affects the lung pericyte population, leading to increased contractility, reduced pulmonary angiogenesis, and aberrant lung development, as observed in CDH.
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