ABSTRACT
Atoh7 has been believed to be essential for establishing the retinal ganglion cell (RGC) lineage, and Pou4f2 and Isl1 are known to regulate RGC specification and differentiation. Here we report our further study of the roles of these transcription factors. Using bulk RNA-seq, we identify genes regulated by the three transcription factors, which expand our understanding of the scope of downstream events. Using scRNA-seq on wild-type and mutant retinal cells, we reveal a transitional cell state of retinal progenitor cells (RPCs) co-marked by Atoh7 and other genes for different lineages and shared by all early retinal lineages. We further discover the unexpected emergence of the RGC lineage in the absence of Atoh7. We conclude that competence of RPCs for different retinal fates is defined by lineage-specific genes co-expressed in the transitional state and that Atoh7 defines the RGC competence and collaborates with other factors to shepherd transitional RPCs to the RGC lineage.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Retina/metabolism , Retinal Ganglion Cells/metabolism , Transcriptome , Animals , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Loss of Function Mutation , Mice , RNA, Small Cytoplasmic , Sequence Analysis , Stem Cells , Transcription Factor Brn-3B/genetics , Transcription Factor Brn-3B/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
BACKGROUND: Cholinergic neuronal loss is one of the hallmarks of AD related neurodegeneration; however, preclinical promise of α7 nAChR drugs failed to translate into humans. CHRFAM7A, a uniquely human fusion gene, is a negative regulator of α7 nAChR and was unaccounted for in preclinical models. METHODS: Molecular methods: Function of CHRFAM7A alleles was studied in vitro in two disease relevant phenotypic readouts: electrophysiology and Aß uptake. Genome edited human induced pluripotent stem cells (iPSC) were used as a model system with the human context. Double blind pharmacogenetic study: We performed double-blind pharmacogenetic analysis on the effect of AChEI therapy based on CHRFAM7A carrier status in two paradigms: response to drug initiation and DMT effect. Mini Mental Status Examination (MMSE) was used as outcome measure. Change in MMSE score from baseline was compared by 2-tailed T-test. Longitudinal analysis of clinical outcome (MMSE) was performed using a fitted general linear model, based on an assumed autoregressive covariance structure. Model independent variables included age, sex, and medication regimen at the time of the first utilized outcome measure (AChEI alone or AChEI plus memantine), APOE4 carrier status (0, 1 or 2 alleles as categorical variables) and CHRFAM7A genotype. FINDINGS: The direct and inverted alleles have distinct phenotypes. Functional CHRFAM7A allele classifies the population as 25% non-carriers and 75% carriers. Induced pluripotent stem cell (iPSC) models α7 nAChR mediated Aß neurotoxicity. Pharmacological readout translates into both first exposure (pâ¯=â¯0.037) and disease modifying effect (pâ¯=â¯0.0048) in two double blind pharmacogenetic studies. INTERPRETATION: CHRFAM7A accounts for the translational gap in cholinergic strategies in AD. Clinical trials not accounting for this uniquely human genetic factor may have rejected drug candidates that would benefit 25% of AD. Reanalyses of the completed trials using this pharmacogenetic paradigm may identify effective therapy.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/therapy , Cholinergic Neurons/metabolism , Recombinant Fusion Proteins , alpha7 Nicotinic Acetylcholine Receptor/genetics , Alleles , Alzheimer Disease/diagnosis , Alzheimer Disease/etiology , Amyloid beta-Peptides/metabolism , Biomarkers , Cell Line , Cholinergic Antagonists/pharmacology , Cholinergic Antagonists/therapeutic use , Drug Evaluation, Preclinical , Fluorescent Antibody Technique , Gene Dosage , Gene Frequency , Genotype , Humans , Induced Pluripotent Stem Cells/metabolism , Phenotype , Synaptic Transmission , Translational Research, Biomedical , Treatment Outcome , alpha7 Nicotinic Acetylcholine Receptor/metabolismABSTRACT
Recently our group demonstrated that acellular tissue engineered vessels (A-TEVs) comprised of small intestinal submucosa (SIS) immobilized with heparin and vascular endothelial growth factor (VEGF) could be implanted into the arterial system of a pre-clinical ovine animal model, where they endothelialized within one month and remained patent. Here we report that immobilized VEGF captures blood circulating monocytes (MC) with high specificity under a range of shear stresses. Adherent MC differentiate into a mixed endothelial (EC) and macrophage (Mφ) phenotype and further develop into mature EC that align in the direction of flow and produce nitric oxide under high shear stress. In-vivo, newly recruited cells on the vascular lumen express MC markers and at later times they co-express MC and EC-specific proteins and maintain graft patency. This novel finding indicates that the highly prevalent circulating MC contribute directly to the endothelialization of acellular vascular grafts under the right chemical and biomechanical cues.
