How epithelial progenitor cells integrate local signals to balance expansion with differentiation during organogenesis is still little understood. Here, we provide evidence that the Hippo pathway effector Yap is a key regulator of this process in the developing lung. We show that when epithelial tubules are forming and branching, a nucleocytoplasmic shift in Yap localization marks the boundary between the airway and the distal lung compartments. At this transition zone, Yap specifies a transcriptional program that controls Sox2 expression and ultimately generates the airway epithelium. Without Yap, epithelial progenitors are unable to properly respond to local TGF-β-induced cues and control levels and distribution of Sox2 to form airways. Yap levels and subcellular localization also markedly influence Sox2 expression and differentiation of adult airway progenitors. Our data reveal a role for the Hippo-Yap pathway in integrating growth-factor-induced cues in the developing and adult lung potentially key for homeostasis and regeneration repair.

Background:The TGF␤ and Hippo pathways are dysregulated in metastatic breast cancers. Results: TGF␤-induced cues and nuclear TAZ/YAP converge at the transcriptional level to control gene expression important for tumorigenesis. Conclusion: TAZ/YAP are required to promote TGF␤-induced tumorigenic phenotypes in breast cancer cells. Significance: Our study reveals novel cross-talk between the TGF␤ pathway and TAZ/YAP in late-stage breast cancers.

Basal cells are multipotent airway progenitors that generate distinct epithelial cell phenotypes crucial for homeostasis and repair of the conducting airways. Little is known about how these progenitor cells expand and transition to differentiation to form the pseudostratified airway epithelium in the developing and adult lung. Here, we show by genetic and pharmacological approaches that endogenous activation of Notch3 signaling selectively controls the pool of undifferentiated progenitors of upper airways available for differentiation. This mechanism depends on the availability of Jag1 and Jag2, and is key to generating a population of parabasal cells that later activates Notch1 and Notch2 for secretory-multiciliated cell fate selection. Disruption of this mechanism resulted in aberrant expansion of basal cells and altered pseudostratification. Analysis of human lungs showing similar abnormalities and decreased NOTCH3 expression in subjects with chronic obstructive pulmonary disease suggests an involvement of NOTCH3-dependent events in the pathogenesis of this condition.

SUMMARY Epithelial cells undergo dynamic polarity changes as organs pattern, but the relationship between epithelial polarity and cell fate is poorly understood. Using the developing lung as a model, we have found that distinct alterations in apical-basal polarity dictate airway epithelial differentiation. We demonstrate that Crb3, a Crumbs isoform that determines epithelial apical domain identity, is required for airway differentiation by controlling the localization of the transcriptional regulator Yap. We show that Crb3 promotes interaction between Yap and the Hippo pathway kinases Lats1/2 at apical cell junctions to induce Yap phosphorylation and cytoplasmic retention, which drives cell differentiation. Loss of Crb3 in developing mouse airways, or in isolated adult airway progenitors, results in unrestricted nuclear Yap activity and consequent cell differentiation defects. Our findings demonstrate that polarity-dependent cues control airway cell differentiation, offering important molecular insight into organ patterning.

Systemic sclerosis (scleroderma, SSc) is a devastating fibrotic disease with few treatment options. Fumaric acid esters, including dimethyl fumarate (DMF, Tecfidera; Biogen, Cambridge, MA), have shown therapeutic effects in several disease models, prompting us to determine whether DMF is effective as a treatment for SSc dermal fibrosis. We found that DMF blocks the profibrotic effects of transforming growth factor-β (TGFβ) in SSc skin fibroblasts. Mechanistically, we found that DMF treatment reduced nuclear localization of transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP) proteins via inhibition of the phosphatidylinositol 3 kinase/protein kinase B (Akt) pathway. In addition, DMF abrogated TGFβ/Akt1 mediated inhibitory phosphorylation of glycogen kinase 3β (GSK3β) and a subsequent β-transducin repeat-containing proteins (βTRCP) mediated proteasomal degradation of TAZ, as well as a corresponding decrease of TAZ/YAP transcriptional targets. Depletion of TAZ/YAP recapitulated the antifibrotic effects of DMF. We also confirmed the increase of TAZ/YAP in skin biopsies from patients with diffuse SSc. We further showed that DMF significantly diminished nuclear TAZ/YAP localization in fibroblasts cultured on a stiff surface. Importantly, DMF prevented bleomycin-induced skin fibrosis in mice. Together, our work demonstrates a mechanism of the antifibrotic effect of DMF via inhibition of Akt1/GSK3β/TAZ/YAP signaling and confirms a critical role of TAZ/YAP in mediating the profibrotic responses in dermal fibroblasts. This study supports the use of DMF as a treatment for SSc dermal fibrosis.

Efficient generation of human induced pluripotent stem cell (hiPSC)-derived human intestinal organoids (HIOs) would facilitate the development of in vitro models for a variety of diseases that affect the gastrointestinal tract, such as inflammatory bowel disease or Cystic Fibrosis. Here, we report a directed differentiation protocol for the generation of mesenchyme-free HIOs that can be primed towards more colonic or proximal intestinal lineages in serum-free defined conditions. Using a CDX2 eGFP iPSC knock-in reporter line to track the emergence of hindgut progenitors, we follow the kinetics of CDX2 expression throughout directed differentiation, enabling the purification of intestinal progenitors and robust generation of mesenchyme-free organoids expressing characteristic markers of small intestinal or colonic epithelium. We employ HIOs generated in this way to measure CFTR function using cystic fibrosis patient-derived iPSC lines before and after correction of the CFTR mutation, demonstrating their future potential for disease modeling and therapeutic screening applications.

Salivary glands, such as submandibular glands (SMGs), are composed of branched epithelial ductal networks that terminate in acini that together produce, transport and secrete saliva. Here, we show that the transcriptional regulator Yap, a key effector of the Hippo pathway, is required for the proper patterning and morphogenesis of SMG epithelium. Epithelial deletion of Yap in developing SMGs results in the loss of ductal structures, arising from reduced expression of the EGF family member Epiregulin, which we show is required for the expansion of Krt5/Krt14-positive ductal progenitors. We further show that epithelial deletion of the Lats1 and Lats2 genes, which encode kinases that restrict nuclear Yap localization, results in morphogenesis defects accompanied by an expansion of Krt5/Krt14-positive cells. Collectively, our data indicate that Yap-induced Epiregulin signaling promotes the identity of SMG ductal progenitors and that removal of nuclear Yap by Lats1/2-mediated signaling is critical for proper ductal maturation.DOI: http://dx.doi.org/10.7554/eLife.23499.001

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