RESUMO
Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.
Assuntos
Blastocisto , Diferenciação Celular , Endoderma , Animais , Endoderma/metabolismo , Endoderma/citologia , Camundongos , Blastocisto/metabolismo , Blastocisto/citologia , Linhagem da Célula , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Transdução de Sinais , Desenvolvimento Embrionário , Janus Quinases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição STAT/metabolismo , Fatores de Transcrição/metabolismo , Feminino , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologiaRESUMO
Splice-switching oligonucleotides (SSOs) are antisense compounds that act directly on pre-mRNA to modulate alternative splicing (AS). This study demonstrates the value that artificial intelligence/machine learning (AI/ML) provides for the identification of functional, verifiable, and therapeutic SSOs. We trained XGboost tree models using splicing factor (SF) pre-mRNA binding profiles and spliceosome assembly information to identify modulatory SSO binding sites on pre-mRNA. Using Shapley and out-of-bag analyses we also predicted the identity of specific SFs whose binding to pre-mRNA is blocked by SSOs. This step adds considerable transparency to AI/ML-driven drug discovery and informs biological insights useful in further validation steps. We applied this approach to previously established functional SSOs to retrospectively identify the SFs likely to regulate those events. We then took a prospective validation approach using a novel target in triple negative breast cancer (TNBC), NEDD4L exon 13 (NEDD4Le13). Targeting NEDD4Le13 with an AI/ML-designed SSO decreased the proliferative and migratory behavior of TNBC cells via downregulation of the TGFß pathway. Overall, this study illustrates the ability of AI/ML to extract actionable insights from RNA-seq data.
Assuntos
Processamento Alternativo , Inteligência Artificial , Aprendizado de Máquina , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/genética , Linhagem Celular Tumoral , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Oligonucleotídeos Antissenso/genética , Movimento Celular/genética , Spliceossomos/metabolismo , Spliceossomos/genética , Oligonucleotídeos/genética , FemininoRESUMO
The emergence of mechanobiology has unveiled complex mechanisms by which cells adjust intracellular force production to their needs. Most communicable intracellular forces are generated by myosin II, an actin-associated molecular motor that transforms adenosine triphosphate (ATP) hydrolysis into contraction in nonmuscle and muscle cells. Myosin II-dependent force generation is tightly regulated, and deregulation is associated with specific pathologies. Here, we focus on the role of myosin II (nonmuscle myosin II, NMII) in force generation and mechanobiology. We outline the regulation and molecular mechanism of force generation by NMII, focusing on the actual outcome of contraction, that is, force application to trigger mechanosensitive events or the building of dissipative structures. We describe how myosin II-generated forces drive two major types of events: modification of the cellular morphology and/or triggering of genetic programs, which enhance the ability of cells to adapt to, or modify, their microenvironment. Finally, we address whether targeting myosin II to impair or potentiate its activity at the motor level is a viable therapeutic strategy, as illustrated by recent examples aimed at modulating cardiac myosin II function in heart disease.