Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ

The ability to induce autologous tissue-specific stem cells in culture could have a variety of applications in regenerative medicine and disease modeling. Here we show that transient expression of exogenous YAP or its closely related paralogue TAZ in primary differentiated mouse cells can induce conversion to a tissue-specific stem/progenitor cell state. Differentiated mammary gland, neuronal, and pancreatic exocrine cells, identified using a combination of cell sorting and lineage tracing approaches, efficiently convert to proliferating cells with properties of stem/progenitor cells of their respective tissues after YAP induction. YAP-induced mammary stem/progenitor cells show molecular and functional properties similar to endogenous MaSCs, including organoid formation and mammary gland reconstitution after transplantation. Because YAP/TAZ function is also important for self-renewal of endogenous stem cells in culture, our findings have implications for understanding the molecular determinants of the somatic stem cell state

A GENE NETWORK FOR HEAD ORGANIZER FORMATION

The Spemann organizer determines the body plan, in its full variety of tissue components, normal proportion and placement. Although individual organizer's inducers responsible for head and trunk inductions have been identified, how these are handed out in perfect quantitative, spatial and temporal coordination remains enigmatic. Here we focused on the mechanisms establishing the organizer own architecture as key to explain such signaling coordination. We found that a crosstalk between two ligands emanating from the trunk organizer, Nodal and ADMP, defines a miniature anteroposterior axis. At its posterior pole, Nodal protects headinduction by competing ADMP for a shared receptor, ACVR2a; ADMP is allowed to signal more anteriorly to restrain these inductions. These opposing signaling centers reciprocally adjust their strength and range of activity by multiple negative and positive loops. In so doing, the levels of head inducers remains proportional to signals generated in the trunk and buffered against their fluctuations. Several new regulatory elements are essential for this dynamic crosstalk, including a microRNA, miR-15/16, endowing robustness to head formation. In sum, we propose a model that offers a molecular explanation for key properties of the Spemann's organizer.L'organizzatore di Spemann è un tessuto con proprietà uniche in quanto determina il piano corporeo dei vertebrati: la totalità dei vari componenti tissutali, nelle giuste proporzioni e corretto posizionamento. Nonostante che i singoli “induttori” di testa e tronco siano stati identificati, il come questi si assemblino in modo coordinato (quantitativamente, spazialmente e temporalmente) rimane tuttora un mistero. Per comprendere questo fenomeno, in questa tesi ci siamo concentrati sui meccanismi che stabiliscono l’architettura interna dello stesso organizzatore. Abbiamo trovato che l’attività di due ligandi, Nodal ed ADMP, realizza una rete genica che definisce un asse antero-posteriore in miniatura, localizzato nell’endoderma dorsale. Al polo posteriore di questo “asse”, Nodal protegge l’induzione della testa competendo con ADMP per un recettore condiviso, ACVR2a; ADMP riesce ad agire solo molto più anteriormente, e nel fare questo, restringe le induzioni di Nodal. Questi segnali opposti riaggiustano la loro forza e dominio di azione in modo reciproco, attraverso feedback negativi e positivi. Così facendo, i livelli di induttori della testa restano proporzionali ai segnali generali dal tronco e tamponati contro eventuali variazioni degli stessi. Nuovi elementi di regolazione sono essenziali in questa dinamica comunicazione; tra questi, un microRNA, miR-15/16, che garantisce robustezza alla formazione della testa. In conclusione, qui proponiamo un modello che offre una spiegazione molecolare delle proprietà dell’organizzatore di Spemann

Convergence of p53 and TGF-beta signaling networks.

p53 is a protein with many talents. One of the most fundamental is the ability to act as essential growth checkpoint that protects cells against cellular transformation. p53 does so through the induction of genes leading to growth arrest or apoptosis. Most of the studies focusing on the mechanisms of p53 activity have been performed in cultured cells upon treatment with well-established p53-activating inputs, such as high doses of radiations, DNA-damaging drugs and activated oncogenes. However, how the tumor suppressive functions of p53 become concerted with the extracellular cues arriving at the cell surface during tissue homeostasis, remains largely unknown. Intriguingly, two recent papers have shed new light into this unexplored field, indicating that p53 plays a key role in TGF-beta-induced growth arrest and, unexpectedly, in the developmental effects of TGF-beta in early embryos. Here we review and comment on these findings and on their implications for cancer biology

Germ-Layer Specification and Control of Cell Growth by Ectodermin, a Smad4 Ubiquitin Ligase

TGF-beta signaling is essential for development and proliferative homeostasis. During embryogenesis, maternal determinants act in concert with TGF-beta signals to form mesoderm and endoderm. In contrast, ectoderm specification requires the TGF-beta response to be attenuated, although the mechanisms by which this is achieved remain unknown. In a functional screen for ectoderm determinants, we have identified Ectodermin (Ecto). In Xenopus embryos, Ecto is essential for the specification of the ectoderm and acts by restricting the mesoderm-inducing activity of TGF-beta signals to the mesoderm and favoring neural induction. Ecto is a RING-type ubiquitin ligase for Smad4, a TGF-beta signal transducer. Depletion of Ecto in human cells enforces TGF-beta-induced cytostasis and, moreover, plays a causal role in limiting the antimitogenic effects of Smad4 in tumor cells. We propose that Ectodermin is a key switch in the control of TGF-beta gene responses during early embryonic development and cell proliferation

Integration of TGF-beta and Ras/MAPK signaling through p53 phosphorylation

During development and tissue homeostasis, cells must integrate different signals. We investigated how cell behavior is controlled by the combined activity of transforming growth factor-beta (TGF-beta) and receptor tyrosine kinase (RTK) signaling, whose integration mechanism is unknown. We find that RTK/Ras/MAPK (mitogen-activated protein kinase) activity induces p53 N-terminal phosphorylation, enabling the interaction of p53 with the TGF-beta-activated Smads. This mechanism confines mesoderm specification in Xenopus embryos and promotes TGF-beta cytostasis in human cells. These data indicate a mechanism to allow extracellular cues to specify the TGF-beta gene-expression program

MicroRNA control of nodal signalling

MicroRNAs are crucial modulators of gene expression, yet their involvement as effectors of growth factor signalling is largely unknown. Ligands of the transforming growth factor-beta superfamily are essential for development and adult tissue homeostasis. In early Xenopus embryos, signalling by the transforming growth factor-beta ligand Nodal is crucial for the dorsal induction of the Spemann's organizer. Here we report that Xenopus laevis microRNAs miR-15 and miR-16 restrict the size of the organizer by targeting the Nodal type II receptor Acvr2a. Endogenous miR-15 and miR-16 are ventrally enriched as they are negatively regulated by the dorsal Wnt/beta-catenin pathway. These findings exemplify the relevance of microRNAs as regulators of early embryonic patterning acting at the crossroads of fundamental signalling cascades

YAP/TAZ Incorporation in the \u3b2-Catenin Destruction Complex Orchestrates the Wnt Response

The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the \u3b2-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for \u3b2-TrCP recruitment to the complex and \u3b2-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/\u3b2-catenin signaling. In line, the \u3b2-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppressio

FAM/USP9x, a Deubiquitinating Enzyme Essential for TGF beta Signaling, Controls Smad4 Monoubiquitination

The assembly of the Smad complex is critical for TGFbeta signaling, yet the mechanisms that inactivate or empower nuclear Smad complexes are less understood. By means of siRNA screen we identified FAM (USP9x), a deubiquitinase acting as essential and evolutionarily conserved component in TGFbeta and bone morphogenetic protein signaling. Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits Smad4 by impeding association with phospho-Smad2. FAM reverts this negative modification, re-empowering Smad4 function. FAM opposes the activity of Ectodermin/Tif1gamma (Ecto), a nuclear factor for which we now clarify a prominent role as Smad4 monoubiquitin ligase. Our study points to Smad4 monoubiquitination and deubiquitination as a way for cells to set their TGFbeta responsiveness: loss of FAM disables Smad4-dependent responses in several model systems, with Ecto being epistatic to FAM. This defines a regulative ubiquitination step controlling Smads that is parallel to those impinging on R-Smad phosphorylation