Clinical relevance of rapid FOXF1-targeted sequencing in patients suspected of alveolar capillary dysplasia with misalignment of pulmonary veins

Alveolar capillary dysplasia with misalignment of pulmonary Veins (ACDMPV) is a lethal congenital lung disorder that presents shortly after birth with respiratory failure and therapy-resistant pulmonary hypertension. It is associated with heterozygous point mutations and genomic deletions that involve the FOXF1 gene or its upstream regulatory region. Patients are unresponsive to the intensive treatment regiments and suffer unnecessarily, because ACDMPV is not always timely recognized and histological diagnosis is invasive and time-consuming. Here, we demonstrate the usefulness of a non-invasive, fast genetic test for FOXF1 variants that we previously developed to rapidly diagnose ACDMPV and reduce the time of hospitalization.</p

Cellular Origin(s) of Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a structural birth defect characterized by a diaphragmatic defect, lung hypoplasia and structural vascular defects. In spite of recent developments, the pathogenesis of CDH is still poorly understood. CDH is a complex congenital disorder with multifactorial etiology consisting of genetic, cellular and mechanical factors. This review explores the cellular origin of CDH pathogenesis in the diaphragm and lungs and describes recent developments in basic and translational CDH research

Generation of a biotinylatable Sox2 mouse model to identify Sox2 complexes in vivo

Sox2 is a Sry-box containing family member of related transcription factors sharing homology in their DNA binding domain. Sox2 is important during different stages of development, and previously we showed that Sox2 plays an important role in branching morphogenesis and epithelial cell differentiation in lung development. The transcriptional activity of Sox2 depends on its interaction with other proteins, leading to ‘complex-specific’ DNA binding and transcriptional regulation. In this study, we generated a mouse model containing a biotinylatable-tag targeted at the translational start site of the endogenous Sox2 gene (bioSox2). This tag was biotinylated by the bacterial birA protein and the resulting bioSox2 protein was used to identify associating partners of Sox2 at different phases of lung development in vivo (the Sox2 interactome). Homozygous bioSox2 mice are viable and fertile irrespective of the biotinylation of the bio tag, indicating that the bioSox2 gene is normally expressed and the protein is functional in all tissues. This suggests that partners of Sox2 are most likely able to associate with the bioSox2 protein. BioSox2 complexes were isolated with high affinity using streptavidin beads and analysed by MALDI-ToF mass spectrometry analysis. Several of the identified binding partners are already shown to have a respiratory phenotype. Two of these partners, Wdr5 and Tcf3, were validated to confirm their association in Sox2 complexes. This bioSox2 mouse model will be a valuable tool for isolating in vivo Sox2 complexes from different tissues

Identification of SOX2 Interacting Proteins in the Developing Mouse Lung With Potential Implications for Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia is a structural birth defect of the diaphragm, with lung hypoplasia and persistent pulmonary hypertension. Aside from vascular defects, the lungs show a disturbed balance of differentiated airway epithelial cells. The Sry related HMG box protein SOX2 is an important transcription factor for proper differentiation of the lung epithelium. The transcriptional activity of SOX2 depends on interaction with other proteins and the identification of SOX2-associating factors may reveal important complexes involved in the disturbed differentiation in CDH. To identify SOX2-associating proteins, we purified SOX2 complexes from embryonic mouse lungs at 18.5 days of gestation. Mass spectrometry analysis of SOX2-associated proteins identified several potential candidates, among which were the Chromodomain Helicase DNA binding protein 4 (CHD4), Cut-Like Homeobox1 (CUX1), and the Forkhead box proteins FOXP2 and FOXP4. We analyzed the expression patterns of FOXP2, FOXP4, CHD4, and CUX1 in lung during development and showed co-localization with SOX2. Co-immunoprecipitations validated the interactions of these four transcription factors with SOX2, and large-scale chromatin immunoprecipitation (ChIP) data indicated that SOX2 and CHD4 bound to unique sites in the genome, but also co-occupied identical regions, suggesting that these complexes could be involved in co-regulation of genes involved in the respiratory system

Sox21 modulates sox2-initiated differentiation of epithelial cells in the extrapulmonary airways

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.</p