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Pulmonary neuroendocrine (NE) cells represent a small population in the airway epithelium, but despite this, hyperplasia of NE cells is associated with several lung diseases, such as congenital diaphragmatic hernia and bronchopulmonary dysplasia. The molecular mechanisms causing the development of NE cell hyperplasia remains poorly understood. Previously, we showed that the SOX21 modulates the SOX2-initiated differentiation of epithelial cells in the airways. Here, we show that precursor NE cells start to develop in the SOX2 + SOX21 + airway region and that SOX21 suppresses the differentiation of airway progenitors to precursor NE cells. During development, clusters of NE cells start to form and NE cells mature by expressing neuropeptide proteins, such as CGRP. Deficiency in SOX2 resulted in decreased clustering, while deficiency in SOX21 increased both the numbers of NE ASCL1 + precursor cells early in development, and the number of mature cell clusters at E18.5. In addition, at the end of gestation (E18.5), a number of NE cells in Sox2 heterozygous mice, did not yet express CGRP suggesting a delay in maturation. In conclusion, SOX2 and SOX21 function in the initiation, migration and maturation of NE cells.

The first breath of air at birth marks the beginning of extrauterine life, and breathing problems due to incomplete lung development or acute respiratory distress are common in premature babies and respiratory diseases. However, the underlying molecular mechanisms remain poorly understood. Here we show that the microtubule plus-end-tracking protein CLASP1 is required for neonatal lung function and survival. CLASP1 is expressed in the lungs and associated respiratory structures throughout embryonic development. Clasp1 disruption in mice caused intrauterine growth restriction and neonatal lethality due to acute respiratory failure. Knockout animals showed impaired lung inflation associated with smaller rib cage formation and abnormal diaphragm innervation. Live-cell analysis of microtubule dynamics in cultured hippocampal neurons revealed an increased catastrophe rate, consistent with a role of CLASP1 in neurite outgrowth. Histological and gene expression studies indicated that CLASP1 is required for normal pneumocyte differentiation and fetal lung maturation. Thus, CLASP1-mediated regulation of microtubule dynamics assists multiple systems essential for neonatal lung function and survival.

Pulmonary neuroendocrine (NE) cells represent a small population in the airway epithelium, but despite this, hyperplasia of NE cells is associated with several lung diseases, such as congenital diaphragmatic hernia and bronchopulmonary dysplasia. The molecular mechanisms causing the development of NE cell hyperplasia remains poorly understood. Previously, we showed that the SOX21 modulates the SOX2-initiated differentiation of epithelial cells in the airways (Eenjes, 2021). Here, we show that precursor NE cells start to develop in the SOX2+SOX21+ airway region and that SOX21 suppresses the differentiation of airway progenitors to precursor NE cells. During development, clusters of NE cells start to form and NE cells mature by expressing neuropeptide proteins, such as CGRP. Deficiency in SOX2 resulted in decreased clustering, while deficiency in SOX21 increased both the numbers of NE ASCL1+ precursor cells early in development, and the number of cell clusters at E18.5. In addition, at the end of gestation (E18.5), a number of NE cells in Sox2 heterozygous mice, did not yet express CGRP suggesting a delay in maturation. In conclusion, SOX2 and SOX21 function in the initiation, migration and maturation of NE cells.

SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe SOX21 patterning of the proximal airway epithelium which coincides with high levels of SOX2. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. We show that loss of SOX21 results in increased differentiation of progenitor cells during murine lung development. SOX21 inhibits SOX2-induced differentiation by antagonizing SOX2 binding on different promotors. SOX21 remains expressed in adult tracheal epithelium and submucosal glands, where SOX21 modulates SOX2-induced differentiation in a similar fashion. Using fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+SOX21+ regionalization is conserved in human. Thus SOX21 modulates SOX2-initiated differentiation in extrapulmonary epithelial cells during development and regeneration after injury.

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.

Esophageal atresia (EA) and tracheoesophageal fistula (TEF) are relatively frequently occurring foregut malformations. EA/TEF is thought to have a strong genetic component. Not much is known regarding the biological processes disturbed or which cell type is affected in patients. This hampers the detection of the responsible culprits (genetic or environmental) for the origin of these congenital anatomical malformations. Therefore, we examined gene expression patterns in the TEF and compared them to the patterns in esophageal, tracheal and lung control samples. We studied tissue organization and key proteins using immunohistochemistry. There were clear differences between TEF and control samples. Based on the number of differentially expressed genes as well as histological characteristics, TEFs were most similar to normal esophagus. The BMP-signaling pathway, actin cytoskeleton and extracellular matrix pathways are downregulated in TEF. Genes involved in smooth muscle contraction are overexpressed in TEF compared to esophagus as well as trachea. These enriched pathways indicate myofibroblast activated fibrosis. TEF represents a specific tissue type with large contributions of intestinal smooth muscle cells and neurons. All major cell types present in esophagus are present—albeit often structurally disorganized—in TEF, indicating that its etiology should not be sought in cell fate specification.

We show that hypoxia inducible factor 2α (HIF2α) is highly expressed in patients with pulmonary hypertension (PH). HIF2α is expressed in every patient with congenital diaphragmatic hernia, while only half of the controls express HIF2α. Our data suggest that HIF2α is a link between hypoxia and the development of PH.

Background Patients with congenital diaphragmatic hernia (CDH) have structural and functional different pulmonary vessels, leading to pulmonary hypertension. They often fail to respond to standard vasodilator therapy targeting the major vasoactive pathways, causing a high morbidity and mortality. We analyzed whether the expression of crucial members of these vasoactive pathways could explain the lack of responsiveness to therapy in CDH patients. Methods The expression of direct targets of current vasodilator therapy in the endothelin and prostacyclin pathway was analyzed in human lung specimens of control and CDH patients. Results CDH lungs showed increased expression of both ETA and ETB endothelin receptors and the rate-limiting Endothelin Converting Enzyme (ECE-1), and a decreased expression of the prostaglandin-I2 receptor (PTGIR). These data were supported by increased expression of both endothelin receptors and ECE-1, endothelial nitric oxide synthase and PTGIR in the well-established nitrofen-CDH rodent model. Conclusions Together, these data demonstrate aberrant expression of targeted receptors in the endothelin and prostacyclin pathway in CDH already early during development. The analysis of this unique patient material may explain why a significant number of patients do not respond to vasodilator therapy. This knowledge could have important implications for the choice of drugs and the design of future clinical trials internationally.

Colorectal cancer (CRC) can be divided into non-mucinous and mucinous subtypes, of which the latter portends to have a worse clinical prognosis. A previous study suggested a putative link between SOX2 expression observed selectively in mucinous CRC and the induction of the gastric mucin MUC5AC. In this study, we re-evaluated the expression behavior of SOX2, MUC5AC, and CDX2 in both types of CRC. We performed immunohistochemical analysis on 90 cases of non-mucinous CRCs, 57 cases of mucinous CRCs, and 15 case-matched normal intestinal mucosa. In contrast to the previously suggested link between SOX2 and mucinous CRC, we observe aberrant expression of SOX2 at equal levels in both subtypes. Fluorescence in situ hybridization (FISH) analysis shows that expression is not attributed to genomic amplification. While SOX2 and CDX2 are normally expressed in a reciprocal manner, SOX2-positive tumor cells co-express CDX2. Furthermore, we show that MUC5AC is expressed independently of SOX2. In conclusion, we show that aberrant SOX2 expression is specifically linked neither to mucinous CRCs nor to the induction of MUC5AC, in contrast to previous suggestions.

Patients with congenital diaphragmatic hernia (CDH) suffer from severe pulmonary hypertension attributable to altered development of the pulmonary vasculature, which is often resistant to vasodilator therapy. Present treatment starts postnatally even though significant differences in the pulmonary vasculature are already present early during pregnancy. We examined the effects of prenatal treatment with the phosphodiesterase-5 inhibitor sildenafil on pulmonary vascular development in experimental CDH starting at a clinically relevant time. The well-established, nitrofen-induced CDH rodent model was treated daily with 100 mg/kg sildenafil from day 17.5 until day 20.5 of gestation (E17.5–20.5). Importantly, this timing perfectly corresponds to the developmental stage of the lung at 20 wk of human gestation, when CDH is detectable by 2D-ultrasonography and/or MRI. At E21.5 pups were delivered by caesarean section and euthanized by lethal injection of pentobarbital. The lungs were isolated and subsequently analyzed using immunostaining, real-time PCR, and volume measurements. Prenatal treatment with sildenafil improved lung morphology and attenuated vascular remodeling with reduced muscularization of the smaller vessels. Pulmonary vascular volume was not affected by sildenafil treatment. We show that prenatal treatment with sildenafil within a clinically relevant period improves pulmonary vascular development in an experimental CDH model. This may have important implications for the management of this disease and related pulmonary vascular diseases in human.

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 2 alpha (Hif2 alpha) 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 Hif2 alpha in Type II cells by conditionally expressing an oxygen-insensitive mutant of Hif2 alpha in airway epithelial cells during development. Newborn mice expressing the mutant Hif2 alpha 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 Hif2 alpha is a key regulator in alveolar maturation and the production of phospholipids by Type II cells.

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 occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF). HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS) or weak transcriptional activators (HIF3α/NEPAS). Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel function of Hif3α beyond the hypoxia response.

The adult lung contains several distinct stem cells, although their properties and full potential are still being sorted out. We previously showed that ectopic Sox2 expression in the developing lung manipulated the fate of differentiating cells. Here, we addressed the question whether fully differentiated cells could be redirected towards another cell type. Therefore, we used transgenic mice to express an inducible Sox2 construct in type II pneumocytes, which are situated in the distal, respiratory areas of the lung. Within three days after the induction of the transgene, the type II cells start to proliferate and form clusters of cuboidal cells. Prolonged Sox2 expression resulted in the reversal of the type II cell towards a more embryonic, precursor-like cell, being positive for the stem cell markers Sca1 and Ssea1. Moreover, the cells started to co-express Spc and Cc10, characteristics of bronchioalveolar stem cells. We demonstrated that Sox2 directly regulates the expression of Sca1. Subsequently, these cells expressed Trp63, a marker for basal cells of the trachea. So, we show that the expression of one transcription factor in fully differentiated, distal lung cells changes their fate towards proximal cells through intermediate cell types. This may have implications for regenerative medicine, and repair of diseased and damaged lungs.

Lung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF). HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS) or weak transcriptional activators (HIF3α/NEPAS). Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel function of Hif3α beyond the hypoxia response.

Background Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterized by the herniation of abdominal organs into the chest cavity. The high mortality and morbidity of CDH patients are primarily caused by the associated pulmonary hypertension (PH), characterized by the thickening of the vascular media and adventitia. The media consist of heterogeneous populations of vascular smooth muscle cells (VSMC), ranging from synthetic to the characteristic contractile cells. VSMCs are influenced by developmental and environmental cues and may play a role in the development of the structural changes observed in CDH patients. Therefore, we hypothesized that the distribution of the VSMC populations may already be different at the origin of CDH development. Methodology We analyzed the protein expression of specific markers associated with synthetic and contractile VSMC phenotypes in human lungs at different developmental stages. Next, we compared lungs of premature and term CDH patients, as well as patients with lung hypoplasia due to renal agenesis or PROM, with age-matched controls. Results Synthetic and contractile VSMCs are distributed in a temporal and spatial specific pattern along the proximodistal axis of the lung. CDH patients have more abundant contractile VSMCs which are also more distally distributed. This different distribution pattern is already observed from 19 weeks of gestation onwards. Conclusion Our data suggest that the more extensive distribution of contractile VSMCs is associated with an early maturation of the pulmonary vasculature, contrasting the concept that CDH might be the result of delayed maturation of the epithelium.

Contains fulltext : 144280.pdf (Publisher’s version ) (Closed access) Congenital gastric-type heteroplasia is common in intestinal duplications and anomalies, which originate from incomplete resorption of the omphalomesenteric duct during development. Two transcription factors determine the proximodistal specification of the gastrointestinal tract, SOX2, expressed exclusively in the proximal part of the primitive gut, and CDX2, expressed solely in the distal part. Aberrant expression of these factors may result in abnormal development and congenital abnormalities. Therefore, we analyzed the expression of SOX2 and CDX2 in a number of pediatric intestinal anomalies. We investigated the expression pattern of SOX2 and CDX2 in three congenital intestinal anomalies in which ectopic gastric tissue may be present, Meckel's diverticulum (N = 8), persistent ductus omphalomesentericus (N = 14), and intestinal duplications (N = 8). CDX2, but not SOX2, was detected in intestinal epithelial cells in tissue lacking gastric heteroplasia. In gastric-type heteroplasia, a reciprocal expression pattern existed between SOX2 and CDX2 in the gastric and intestinal tissues, respectively. Interestingly, patches of CDX2-positive cells were present within the gastric mucosa in a subset of Meckel's diverticula and intestinal duplications, suggesting that it is not the absence of CDX2, but rather the ectopic expression of SOX2 that leads to gastric tissue in the prospective intestinal tissue. This is in concordance with our previous mouse studies. Collectively, our data indicate that a fine balance between SOX2 and CDX2 expression in the gastrointestinal tract is essential for proper development and that ectopic expression of SOX2 may lead to malformations of the gut. 01 mei 2013

Pulmonary hypertension is responsible for significant mortality and morbidity among newborns and infants. The pathology is characterized by pulmonary vascular remodeling with medial hypertrophy and adventitial thickening, leading to decreased gas exchange. Since it is unknown if these abnormalities are reversible, we analyzed these vascular changes in pulmonary hypertensive rats. Exposure of rats to hypobaric hypoxia for 4 weeks induced clinical signs of pulmonary hypertension, such as increased right ventricular systolic pressure, increased right ventricular weight and considerable pulmonary vascular remodeling. The vascular changes were associated with the expression of Non -Muscle Myosin Heavy Chain B in the pre-acinar vessels and an increased expression of alpha Smooth Muscle Actin, Smooth Muscle Myosin Heavy Chain 2 and Calponin in the intra-acinar vessels. The right ventricular systolic pressure and right ventricular weight gradually decreased after specific periods of recovery in normoxia, although this reversal did not reach baseline levels after six weeks at normoxia. However, the cellular changes in the pulmonary vasculature were completely reversed. Development of pulmonary hypertension is associated with an increase of synthetic perivascular cells in the pre-acinar arteries and an aberrant differentiation of perivascular cells in the smallest intra-acinar arteries. These cellular and structural changes in the pulmonary vasculature are completely reversible after recovery in normoxia.