(H) Decreased expression levels of epithelial differentiation markers afterEya1overexpression in MLE-15 cells in culture. birth. Treatment ofEya1-/-epithelial explants in culture with recombinant Fgf10 stimulates epithelial branching. Since Shh expression and activity Cxcr4 are abnormally increased inEya1-/-lungs, we tested whether genetically lowering Shh activity could rescue theEya1-/-lung phenotype. Indeed, genetic reduction of Shh partially rescuesEya1-/-lung defects while restoringFgf10expression. This study provides the first evidence thatEya1regulates Shh signaling in embryonic lung, thus ensuring the proper level of proliferation and differentiation along the proximodistal axis of epithelial, mesenchymal and endothelial cells. These findings uncover novel functions for Eya1 as a critical upstream coordinator of Shh-Fgf10 signaling during embryonic lung development. We conclude, therefore, that Eya1 function is critical for proper coordination of lung epithelial, mesenchymal and vascular development. Keywords:lung development, Eya1, proliferation, differentiation, Shh, Fgf10 == INTRODUCTION == The development of mammalian lung begins when two primary buds consisting of an inner epithelial layer surrounded by mesenchyme arise from the larygotracheal groove in the ventral floor of the primitive foregut. These buds undergo stereotypic rounds of branching and outgrowth to give rise to a tree-like respiratory organ which contains different specialized epithelial cell types organized along Diazepinomicin the proximodistal axis (Cardoso, 2000; Metzger et al, 2009;Warburton et al., 2000,2008). Controlled and rapid proliferation of lung epithelial progenitors, in association with the outgrowth and branching of the epithelial tubes, is essential for producing an alveolar gas diffusion surface sufficient to sustain extra-uterine life. The undifferentiated epithelial cells leave the progenitor pool to give rise first to lineage-committed precursor cells and eventually to fully differentiated cell lineages (Cardoso and L, 2006;Rawlins and Hogan, 2006). Developmental defects in this progression lead to defective differentiation and postnatal respiratory distress (i.e. pulmonary hypoplasia), a hallmark of congenital lung diseases. Epithelial-mesenchymal interactions are crucial to the formation and patterning of the mammalian lung. The pulmonary mesenchyme gives rise to several different cell types, including smooth muscle cells of the upper airways during branching morphogenesis. Moreover, the mesenchyme produces many important growth factors and signaling molecules required for airway epithelial branching and development, e.g. members of the fibroblast growth factor (Fgf) family (reviewed byWarburton et al., 2000,2005,Morrisey and Hogan, 2010). In turn, the epithelium produces signaling molecules essential for mesenchymal proliferation and differentiation including sonic hedgehog (Shh) and bone morphogenetic protein 4 (BMP4) (Bellusci et al., 1996,1997a,b;Litingtung et al., 1998;Pepicelli et al., 1998). In mammals,Eya(Eya1-4) and sine oculis (Six)genes are co-expressed and exhibit synergistic genetic interactions to regulate the development of multiple organs by controlling cell cycle regulators and inhibition of apoptosis (Xu et al., 1997b;Ford et al., 1998;Coletta et al., 2004). BothEya1-/-andSix1-/-mouse embryos have defects in the proliferation and survival of the precursor cells of multiple organs and die at birth (Xu et al., 1999,2002;Li et al., 2003;Zou et al., 2004). Mutations in the Eya1 protein, which is endowed with a phosphatase activity (Li et al., 2003;Jemc and Rebay, 2007) are responsible for Branchio-Oto-Renal and Oto-Facio-Cervical syndromes (Abdelhak et al., 1997;Estefania et al., 2005).Eya1encodes a transcription coactivator containing a divergent N-terminal activation domain and a conserved C-terminal Eya domain that mediates proteinprotein interactions with Six proteins (Xu et al., 1997a,b;Pignoni et al., 1997). The phosphatase function of Eya1 switches Six1 function from repression to activation in the nucleus causing transcriptional activation through recruitment of co-activators providing a mechanism for activation of specific gene targets including those regulating precursor cell proliferation/survival during organogenesis (Li et al., 2003). Yet, the specific functions of Eya1 in lung development are unknown. Herein, we identify a functional role for mammalian Eya1 in lung epithelial development. The lungs ofEya1-/-mice are severely hypoplastic with greatly reduced epithelial branching and increased mesenchymal cellularity and show extensive hemorrhage at birth.Eya1-/-lung epithelial cells show loss of progenitor cell markers, with increased expression of differentiation markers and cell cycle exit. In addition,Eya1-/-embryos and neonates show vascular smooth muscle defects, which lead to vessel rupture and hemorrhage. Eya1-/-lung defects are partly attributed to increased expression and activity of Shh signaling. Furthermore, partial inactivation of theShhgene inEya1-/-lungs partially rescues the epithelial, mesenchymal and endothelial Diazepinomicin defects. == MATERIALS AND METHODS == == Animals and genotyping == Eya1 knockout (KO) mice on the 129/SvEv background andFgf10LacZ/+mice have been published previously (Xu et al., 1999,De Moerlooze et al., 2000,Mailleux et al., 2005).Shhf/fmice were from JAX andSpc-rtTA+/-mice were provided by Dr J. Whitsett (Perl et al., 2002).Eya1+/-;Shhf/WTfemale mice were generated by intercrossingEya1+/-mice with theShhf/fmouse line.Eya1+/–Spc-rtTA+/–tet(o) Cre+/-mice were generated by intercrossingEya1+/-mice with Diazepinomicin theSpc-rtta+/–tet(o) Cre+/+mouse line previously generated in our laboratory. The resultingEya1+/–Spc-rtTA+/–tet(o) Cre+/-mouse males were intercrossed withEya1+/-;Shhf/WTfemales to.
