Alessi, M.-C. | bioRxiv Science

bioRxiv2022

Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia

Expansion of human megakaryoblasts from peripheral blood-derived CD34+ cells is commonly used to characterize inherited or acquired thrombocytopenia and evaluate defects in megakaryocyte (MK) differentiation, MK maturation and proplatelet formation. We applied single-cell RNA sequencing to understand local gene expression changes during megakaryopoiesis (days 6 and 11 of differentiation) in peripheral CD34+ cells from healthy controls and patients with ETV6-related thrombocytopenia. Analysis of gene expression and regulon activity revealed distinct clusters partitioned into seven major cell stages: hematopoietic stem/progenitor cells (HSPC), common-myeloid progenitors (CMP), MK-primed CMP, granulocyte-monocyte progenitors, megakaryocyte-erythroid progenitors (MEP), MK progenitor /mature MK (MKP/MK) and platelets. We observed a subpopulation of MEP that arose directly from HSPC, deviating from the canonical MK differentiation pathway. ETV6 deficiency was characterized by an increase in HSPC, a decrease in MKP/MK, and a lack of platelets. ETV6 deficiency also led to the development of aberrant MEP and MKP/MK cell populations. Genes involved in "mitochondrial" and "DNA repair" pathways were downregulated, while genes involved in "translation" pathways were upregulated. Analysis of patient samples and hematopoietic cell lines transduced with an ETV6 variant revealed increased translation in MK. Ribosomal protein small 6 (RPS6) levels in MK, platelets and peripheral blood mononuclear cells was consistent with the translation findings. Our results provide a framework to understand peripheral CD34+ cell-derived megakaryocytic cultures. Our observations also shed light on ETV6-variant pathology and reveal potential targets for diagnostic and therapeutic purposes. Key points- scRNAseq gain insight into in vitro megakaryopoiesis, identify MK-primed CMP, and a differentiation trajectory that bypasses the CMP. - ETV6 variants led to the development of aberrant MEP and MK cell populations.

bioinformatics↗

bioRxiv2019

Mutations in CalDAG-GEFI Lead to Striatal Signaling Deficits and Psychomotor Symptoms in Multiple Species Including Human

Syndromes caused by mutations in Ras-MAP kinase signaling molecules are known as RASopathies and share features such as developmental delay, autistic traits, and cancer. Syndromic features of Rap-MAP kinase signaling defects remain undefined. CalDAG-GEFI is a calcium-responsive Rap-GTPase activator that is enriched in the matrix of the sensorimotor striatum and down-regulated in Huntingtons disease. We show here that CalDAG-GEFI mutations, including striatum-specific deletions and spontaneous mutations in the enzymatic domain, are associated with psychomotor phenotypes in humans, dogs and mice. The identification of these neural mutants was guided by the overt bleeding phenotype in CalDAG-GEFI knockout mice, and then in humans and other species with conserved platelet signaling deficits. Knockout mice exhibit loss of striatal long-term potentiation and deficits in dopamine, acetylcholine and glutamate signaling, along with delayed motor learning and drug-induced perseverative behaviors. Thus, loss of CalDAG-GEFI signaling produces an evolutionarily conserved syndrome characterized by bleeding and psychomotor dysfunction.

neuroscience↗