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BACKGROUND: Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM. RESULTS: In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics. CONCLUSIONS: In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN.

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2+ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as KrasG12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2+ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and KrasG12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics.

Aging is a complex multifactorial process associated with epigenome dysregulation, increased cellular senescence, and decreased rejuvenation capacity. Short-term cyclic expression of octamer-binding transcription factor 4 (Oct4), sex-determining region Y-box 2 (Sox2), Kruppel-like factor 4 (Klf4), and cellular myelocytomatosis oncogene (cMyc) (OSKM) in wild-type mice improves health but fails to distinguish cell states, posing risks to healthy cells. Here, we delivered a single dose of adeno-associated viruses (AAVs) harboring OSK under the control of the cyclin-dependent kinase inhibitor 2a (Cdkn2a) promoter to specifically partially reprogram aged and stressed cells in a mouse model of Hutchinson-Gilford progeria syndrome (HGPS). Mice showed reduced expression of proinflammatory cytokines and extended life spans upon aged cell–specific OSK expression. The bone marrow and spleen, in particular, showed pronounced gene expression changes, and partial reprogramming in aged HGPS mice led to a shift in the cellular composition of the hematopoietic stem cell compartment toward that of young mice. Administration of AAVs carrying Cdkn2a-OSK to naturally aged wild-type mice also delayed aging phenotypes and extended life spans without altering the incidence of tumor development. Furthermore, intradermal injection of AAVs carrying Cdkn2a-OSK led to improved wound healing in aged wild-type mice. Expression of CDKN2A-OSK in aging or stressed human primary fibroblasts led to reduced expression of inflammation-related genes but did not alter the expression of cell cycle–related genes. This targeted partial reprogramming approach may therefore facilitate the development of strategies to improve health and life span and enhance resilience in the elderly. Editor's summary: Partial cellular reprogramming via cyclic expression of octamer-binding transcription factor 4, sex-determining region Y-box 2, Kruppel-like factor 4, and cellular myelocytomatosis oncogene (OSKM) improves health and life span in mouse models but may lead to tumor induction or organ damage. Here, Sahu and colleagues used adeno-associated viruses to deliver OSK under the control of the cyclin-dependent kinase inhibitor 2a (Cdkn2a) promoter to specifically target stressed and senescent cells in a mouse model of Hutchinson-Guilford progeria syndrome. Treatment extended life spans and improved overall fitness, associated with a shift in the transcriptome toward that seen in younger mice. Similar effects occurred in aged wild-type mice and treatment did not increase tumor occurrence, suggesting that targeting partial cellular reprogramming to specific cell populations may be a more viable rejuvenation strategy moving forward. —Melissa L. Norton [ABSTRACT FROM AUTHOR]

Author(s): Yasuhiko Kawakami [1, 2]; Ãngel Raya [1, 2]; R. Marina Raya [1]; Concepción Rodríguez-Esteban [1]; Juan Carlos Izpisúa Belmonte (corresponding author) [1] The body plan of vertebrates has distinct [...]

Author(s): Ãngel Raya [1, 4]; Yasuhiko Kawakami [1, 4]; Concepción Rodríguez-Esteban [1, 4]; Marta Ibañes [1]; Diego Rasskin-Gutman [1]; Joaquín Rodríguez-León [2]; Dirk Büscher [1]; José A. Feijó [2, 3]; [...]

Three WNT factors have been shown to signal through beta-catenin function as critical regulators of the FGF-8/FGF-10 loop. Induction of Fgf-8 in the limb ectoderm by FGF-10 is mediated by the induction of Wnt-a. Three WNT signals mediated by beta-catenin control both limb initiation and apical ectodermal ridge (AER) induction in the vertebrate embryo.

Two members of the T-box gene family, Tbx4 and Tbx5 are specifically expressed in the hindlimb and forelimb buds respectively. They are shown to be necessary regulators of limb outgrowth, closely linked to the activities of three signalling proteins needed for limb outgrowth and patterning, fibroblast growth factor, bone morphogenetic protein and Wnt. It is also shown that Tbx4 and Tbx5 are involved in the control of limb identity.

Pitx1 gene-deleted mice have reciprocal abnormalities of one dorsal and two ventral anterior pituitary cell types. The abnormalities likely are based on its synergistic functions with other transcription factors and defects in the derivatives of the first brachial arch. The derivatives would includ cleft palate, which would suggest a proliferative defect analogous to that seen in the hindlimb.

The bicoid-related homeodomain transcription factor Pitx2 could be the regulatory force behind the gene network responsible for the morphological events that lead to left-right asymmetries in vertebrates. Pitx2 could interpret and later execute the left-right developmental program dictated by upstream signalling molecules. Pitx2 expression is initiated at the onset of organogenesis and maintained throughout embryogenesis. Its left-right instructive role is required both for the heart and for correct situs of the gut and body rotation.

The radical fringe gene (R-fng) in vertebrates, which encodes a protein called fringe, plays an essential role in vertebrate limb outgrowth. The gene directs the formation and positioning of the apical ectodermal ridge at the dorsoventral limb boundary. This was concluded following the cloning of the chick gene, which is homologous to the Drosophila fringe protein gene. It was also examined whether R-fng expression regulates or is regulated by the genese Wnt-7a and En-1 that establish the dorsoventral boundary of the vertebrate limb. Findings indicate that correct R-fng expression requires the prior establishment of the boundary, but does not influence it.

Research has been conducted on left-right asymmetry establishment during vertebrate embryogenesis. The authors report the existence of the mechanism which involves Notch activity translating initial symmetry breaking event into asymmetric gene expression.

Identification of the precise molecular pathways involved in oncogene-induced transformation may help us gain a better understanding of tumor initiation and promotion. Here, we demonstrate that SOX2+ foregut epithelial cells are prone to oncogenic transformation upon mutagenic insults, such as KrasG12D and p53 deletion. GFP-based lineage-tracing experiments indicate that SOX2+ cells are the cells-of-origin of esophagus and stomach hyperplasia. Our observations indicate distinct roles for oncogenic KRAS mutation and P53 deletion. p53 homozygous deletion is required for the acquisition of an invasive potential, and KrasG12D expression, but not p53 deletion, suffices for tumor formation. Global gene expression analysis reveals secreting factors upregulated in the hyperplasia induced by oncogenic KRAS and highlights a crucial role for the CXCR2 pathway in driving hyperplasia. Collectively, the array of genetic models presented here demonstrate that stratified epithelial cells are susceptible to oncogenic insults, which may lead to a better understanding of tumor initiation and aid in the design of new cancer therapeutics. [ABSTRACT FROM AUTHOR]

Asymmetric expression of Sonic hedgehog (Shh) in Hensen's node of the chicken embryo plays a key role in the genetic cascade that controls left-right asymmetry, but its involvement in left-right specification in other vertebrates remains unclear. We show that mouse embryos lacking Shh display a variety of laterality defects, including pulmonary left isomerism, alterations of heart looping, and randomization of axial turning. Expression of the left-specific gene Lefty-1 is absent in [Shh.sup.-/-] embryos, suggesting that the observed laterality defects could be the result of the lack of Lefty-1. We also demonstrate that retinoic acid (RA) controls Lefty-1 expression in a pathway downstream or parallel to Shh. Further, we provide evidence that RA controls left-right development across vertebrate species. Thus, the roles of Shh and RA in left-right specification indeed are conserved among vertebrates, and the Shh and RA pathways converge in the control of Lefty-1.