The role of Sox2 in lung development:
The lung is a complex, highly organized organ and absolutely required for life. Development of the bronchial tree relies on the intimate interaction between endoderm-derived airway epithelium and the surrounding mesenchyme through a process called “branching morphogenesis”. The last two decades a number of research groups have shown the involvement of a diverse set of genes in this initial process. Recently, we demonstrated that the HMG-box transcription factor Sox2 is mandatory for correct branching of the airways and is important in determining the epithelial cell fate. Using inducible transgenic mice, we showed that Sox2 triggers intrinsic cellular changes leading to the development of abnormal gas exchange surfaces. Although terminal differentiation of airway epithelial cells was not inhibited, it appeared that maintenance of Sox2 expression caused cells to become irresponsive to the branch-inducing signaling molecule Fgf10, which was corroborated by the absence of pERK1/2 and the presence of pSMAD1/5/8. Furthermore, Sox2 maintained cells in a committed precursor-like stage of differentiation, like neuroendocrine cells and basal cells, as shown in the embryonal Sox2-overexpressing lungs. Preliminary data revealed that members of the Notch signaling pathway are expressed in the affected lungs, indicating that Sox2 positive cells influence surrounding cells by cell-cell interactions. These cellular changes induced by aberrant Sox2 expression in epithelial cells eventually resulted in lungs with severely reduced number of airways branches. The few airways present were abnormally enlarged, cyst-like airspaces with aberrant cell types. These structures displayed a proximal airway phenotype, probably caused by an overgrowth of terminal bronchioli.

Current research is focused on the elucidation of the specific pathways influenced by Sox2.

Sox2 overexpression leads to abnormal primitive alveolar formation. External appearances of control (E), heterozygous (F) and homozygous Sox2 (G) overexpressing lungs displaying enlarged airspaces and a marked decrease in the number of airways. Low and high power images of sections stained with hematoxylin and eosin from normal (H, K), heterozygous (I, L) and homozygous transgenic lungs (J, M). Note the undifferentiated dysplastic epithelium in the double transgenic (K–M, arrowhead). The sharp boundary between bronchioles and primitive alveoli in wild-type (K, arrowhead) is absent in the transgenic lungs (L, M). Scale bars: 500 μm (H–J), 50 μm (K–M).

Sox2 overexpression results in the increase in neuroepithelial cells and (pre-) basal cells. Staining of cGRP (A–C), p63 (D–F) andmyc epitope (C, F) in lungs at E18.5 from control (A, D) and Sox2 overexpressing mice (B, C, E, F). cGRP positive staining (neuroepithelial cells) was only detected at the bronchoalveolar ducts in controls (A, arrowheads), and p63 (basal cells) was only expressed in the basal cell layer of the trachea (D, inset). Increased staining for cGRP (B) and p63 (E) was detected in the abnormal airway epithelium expanding into the enlarged airspaces. Note that cGRP positive cells are negative for the Sox2 transgene (C), while Sox2 transgenic expression colocalizes with p63 (E, F). Note that there is no myc-epitope expression in the control lungs (C, insert). Scale bars: 100 μm.

Sox2 and the development of the gut:
The expression pattern of Sox2 in the primitive gut is strictly regulated and exclusively expressed in the anterior part. Its expression appears to be reciprocal to the expression of Cdx2, a marker for the intestinal region of the gut. We are interested to investigate the balance between these two factors, and what is regulating their expression pattern.  
Furthermore, we are surveying different gut associated tumors to evaluate the role of Sox2 and related genes in the occurrence of tumors. Several reports have correlated Sox2 expression with certain tumor types, including tumors of the gastro-intestinal tract. Colorectal carinoma (CRC) is a major health problem and the second cancer related cause of death in The Netherlands. Every year around 10,000 persons are newly diagnosed with CRC. Colorectal carcinoma is primarily associated with older adults and can be roughly divided into non-mucinous (80-90 %) and mucinous (10-20 %) subtypes. It has been hypothesized that serrated polyps and villous adenomas are precursors for mucinous and signet ring cell cancers. The poor prognosis in adults with mucinous CRC, warrants further investigation in early detection and prediction of clinical course. Sox2 has been retrospectively correlated with mucinous CRC, but it is still questionable whether Sox2 expression is essential for initiating a mucinous differentiation pattern and whether it is a good prognostic marker or not. Therefore, we intend to investigate the role of Sox2 in the development of (mucinous) intestinal tumor formation in mice, and to correlate our mouse data with human pathological samples.

Transgene expression in the developing intestine. Cross section through embryonal intestine at 18 dpc after 8 days of doxycycline induction. Immunostaining with an antibody against the myc epitope present within the Sox2 transgene reveals strong positive nuclear staining in intestinal epithelial cells (b). In wild type or single transgenic littermates only background staining is observed, mainly in the cytoplasm (a).

The role of Hif2 α in lung development: 
 Congenital abnormalities of the lung may be envisaged as deranged development. In order to understand the etiology and progression of these type of disorders, it is mandatory to understand the basic principles of normal and abnormal development. As the lungs develop, they prepare to play a major role in the gas exchange between the body and the oxygen-rich atmosphere. However, during the fetal period they grow and differentiate in a relative hypoxic environment. A key player in the oxygen-sensing molecular biology is the Hypoxia Inducible Factor (HIF), which is a heterodimer of a constitutively expressed ARNT/Hif1β subunit, and an oxygen sensitive α subunit, of which three members are currently known (1α, 2α and 3α). Under normoxic conditions, the Hif proteins are hydroxylated and rapidly degraded. However, under hypoxic condition, the proteins are stable and able to transactivate genes that contain a Hypoxic Responsive Element (HRE) in their promoter region. Genes that are under hypoxic regulation are involved in angiogenesis and metabolism. The importance of these factors in lung development is supported by their involvement in diseases like pulmonary hypertension, bronchopulmonary dysplasia and alveolar capillary dysplasia.

Pulmonary arterial hypertension (PAH) of the newborn is a potentially fatal disease of unknown cause and pathology shows a progressive thickening of the media and adventitia of the pulmonary arteries, causing pulmonary hypertension and the subsequent ventilation-perfusion mismatch causes hypoxemia. Current therapy is a matter of trial and error, since PAH is a multifactorial disease. Recently, it was shown that one of the Hif subunits, Hif2α, may be involved in the development of pulmonary hypertension. In addition, analysis of Hif2α expression levels in normal human development showed an increase during gestation, indicating a possible important role for this subunit.

Current research is focused on the role of Hif2α in normal and abnormal development of the lung. Therefore, we make use of a generated conditional overexpressing Hif2α mouse, as well as a conditional Hif2α knockout mouse.

External appearances of control (A) and Hif2α (B) overexpressing lungs show that Hif2α overexpression during embryonal development leads to alveolar abnormalities. Analysis of the alveolar region revealed enlarged, myc-epitope postive type II pneumocytes in the Hif2α overexpressing lungs (D) as compared to controls (C).

The role of vascular smooth muscle cells in pulmonary hypertension
Pulmonary hypertension of the newborn is characterized by a failure of a normal transition to a postnatal circulation and persistence and/or augmentation of anatomical changes in the pulmonary vasculature, called vascular remodeling. Pulmonary hypertension can occur isolated without underlying anomaly or in association with abnormal lung development such as in congenital diaphragmatic hernia and other forms of pulmonary hypoplasia. The vascular remodelling is characterized by a thickening of the vessel wall, as well as changes in the molecular composition, caused by proliferation of smooth muscle cells (SMC) in the media and fibroblasts in the adventitia, recruitment of adventitial cells into the media or circulating cells into the vessel wall and deposition of extracellular matrix proteins. This vascular remodeling is influenced by both (epi)-genetic and environmental factors. Vascular smooth muscle cells perform contractile and synthetic functions, which are associated with different phenotypes. The elongated, spindle-shaped contractile SMCs are essential for managing the luminal diameter by contraction and relaxation, whereas the cobblestone-like synthetic SMCs synthesize large amounts of extracellular matrix components and have increased proliferation and migration capacities. Switching between these phenotypes, called phenotypic modulation, can occur and is thought to determine the functional vascular response.
Current research is focused on the role of the phenotypic modulation of SMCs in the development of pulmonary hypertension. Therefore, the molecular mechanisms involved in pulmonary arterial SMC (PASMC) phenotype heterogeneity are explored using the nitrofen-induced rat CDH model. The PASMCs will be characterized both during normal and abnormal development in the rat and human. Furthermore, putative treatment modalities will be tested on PASMCs. This may contribute to a better treatment of pulmonary vascular disease in infants with pulmonary hypertension.

Differences between rat embryonic lungs, E21, from control (A, C) and Nitrofen-induced CDH (B, D) revealed by anti-smooth muscle actin staining (A, B) and primary PASMCs (C, D)