Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors

The Notch protein is one of the most mechanistically direct transmembrane receptors—the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus, multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues

Parsing patterns: emerging roles of tissue self-organization in health and disease

Patterned morphologies, such as segments, spirals, stripes, and spots, frequently emerge during embryogenesis through self-organized coordination between cells. Yet, complex patterns also emerge in adults, suggesting that the capacity for spontaneous self-organization is a ubiquitous property of biological tissues. We review current knowledge on the principles and mechanisms of self-organized patterning in embryonic tissues and explore how these principles and mechanisms apply to adult tissues that exhibit features of patterning. We discuss how and why spontaneous pattern generation is integral to homeostasis and healing of tissues, illustrating it with examples from regenerative biology. We examine how aberrant self-organization underlies diverse pathological states, including inflammatory skin disorders and tumors. Lastly, we posit that based on such blueprints, targeted engineering of pattern-driving molecular circuits can be leveraged for synthetic biology and the generation of organoids with intricate patterns

The sound of silence:Transgene silencing in mammalian cell engineering

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.ISSN:2405-472

NEGATIVE CONTROL OF SMAD ACTIVITY PATTERNS THE MAMMALIAN EMBRYO

The definition of embryonic potency and induction of specific cell fates are intimately linked to the tight control over TGFβ signaling. Although extracellular regulation of ligand availability has received considerable attention in recent years, surprisingly little is known on the intracellular factors that negatively control Smad activity in mammalian tissues. By means of genetic ablation, here we show that the Smad4 inhibitor Ectodermin (Ecto, also known as TRIM33 or Tif1γ) is required to allow Nodal morphogenetic properties in early mouse embryo. Loss of Ecto invariably drives Nodal responsiveness to the highest, Smad4-dependent threshold of activity; new phenotypes, linked to excessive Nodal activity, emerge from such a modified landscape of Smad responsiveness in both embryonic and extraembryonic territories. In extraembryonic endoderm, Ecto is required to confine expression of Nodal antagonists to the Anterior Visceral Endoderm. In trophoblast cells, Ecto precisely doses Nodal activity, balancing stem cell self-renewal and differentiation. Epiblast-specific Ecto deficiency shifts mesoderm fates toward Node/Organizer fates, revealing the requirement of Smad4 inhibition for the precise allocation of cells along the primitive streak. This study unveils that intracellular control of Smad function by Ecto/Tif1γ is an integral component of how cells read TGFβ signals.La regolazione della pluripotenza e l'induzione di specifici percorsi di differenziamento cellulare sono intimamente connessi a uno stretto controllo della via di segnalazione del TGFβ. La regolazione della parte extracellulare di questa via, cioè quella che concerne la biodisponibilità del ligando, è stata oggetto di numerosi studi in anni recenti. Per quanto riguarda invece la parte intracellulare, pochissimo è noto sull’importanza di fattori che controllino in modo negativo l’attività delle Smad nelle cellule dei tessuti di mammifero. Tramite ablazione genetica, con questo studio dimostriamo come l’inibizione di Smad4 da parte di Ectodermin/Tif1γ/TRIM33 (Ecto) sia richiesta per permettere a Nodal (il ligando TGFβ per eccellenza durante lo sviluppo embrionale mammifero) di svolgere le sue funzioni morfogenetiche durante l’embriogenesi murina. La delezione di Ecto sposta le risposte a Nodal sulla finestra di responsività massima, Smad4 dipendente. In questa situazione di ipereccitata responsività alla segnalazione da parte di Smad, emergono fenotipi nuovi sia in tessuti embrionali che extraembrionali, che sono legati ad un’eccessiva attività di Nodal. Nell’endoderma extraembrionale, Ecto serve per confinare l’espressione degli antagonisti di Nodal nell’endoderma viscerale anteriore. Nelle cellule del trofoblasto, Ecto dosa in modo preciso l’attività di Nodal, permettendo l’instaurarsi del delicato equilibrio tra crescita staminale e differenziamento. La delezione di Ecto specificamente nell’epiblasto incanala il differenziamento del mesoderma verso destini di nodo/tessuto organizzatore, mostrando come sia necessaria un’inibizione di Smad4 per permettere l’allocazione ordinata di cellule ai vari destini lungo la stria primitiva. Questo lavoro dimostra come il controllo negativo sulle funzioni delle Smad da parte di Ecto sia una componente fondamentale del meccanismo di lettura da parte delle cellule dei segnali TGFβ

Tissue Patterning: The Winner Takes It All, the Losers Standing Small

A new mechanism for the selection and differentiation of a single cell, based on mechanical competition with its neighbors and differential TAZ activity, is shown to be at play during zebrafish oogenesis to prevent polyspermy

TGF-beta signaling: Quantitative cues, qualitative outputs

The assembly of the Smad complex is critical for TGFb 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 (DUB) acting as essential and evolutionarily conserved component in TGFb and BMP 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/Tif1g (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 TGFb 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

Programming self-organizing multicellular structures with synthetic cell-cell signaling

A common theme in the self-organization of multicellular tissues is the use of cell-cell signaling networks to induce morphological changes. We used the modular synNotch juxtacrine signaling platform to engineer artificial genetic programs in which specific cell-cell contacts induced changes in cadherin cell adhesion. Despite their simplicity, these minimal intercellular programs were sufficient to yield assemblies with hallmarks of natural developmental systems: robust self-organization into multidomain structures, well-choreographed sequential assembly, cell type divergence, symmetry breaking, and the capacity for regeneration upon injury. The ability of these networks to drive complex structure formation illustrates the power of interlinking cell signaling with cell sorting: Signal-induced spatial reorganization alters the local signals received by each cell, resulting in iterative cycles of cell fate branching. These results provide insights into the evolution of multicellularity and demonstrate the potential to engineer customized self-organizing tissues or materials

Synthetic biology and tissue engineering: toward fabrication of complex and smart cellular constructs

Tissue engineering approaches, with the goals of replacing or recovering damaged or diseased tissues, or of reconstituting tissues in vitro for disease modeling and drug development, have the potential to make significant contributions to medicine. Advances in stem cell biology, biomaterial synthesis and characterization, and microscale technologies have made engineered tissues a reality. However, the classic tools used to build tissues in the lab do not allow for complete control of cell behaviors. More recently, synthetic biology principles have developed robust and versatile approaches to program cells with artificial genetic circuits, where cell behavior and function can be manipulated. At the interface between synthetic biology and tissue engineering, there is space for a new area of investigation where material engineering and cellular engineering complement and sustain each other. In this progress report, synthetic biology principles and how they have been used to engineer cells with potential to dictate cell behavior and function in tissue constructs of the future are briefly described. It is believed that this research area still needs further exploration to fully exploit synthetic biology to make smart and functional cellular constructs for therapeutic and in vitro applications