Project description:Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra-cellular architectures. The role of cytokines in the regulation of HSC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here we studied the early commitment of human HSC and identified specific microtubule network rearrangements and nucleus shape changes that accompany cells differentiation toward the myeloid lineage. This work established the role of microtubules in the mechanical regulation of nucleus shape, chromatin architecture and HSC differentiation and open new perspectives in our appreciation of the implication of intra-cellular forces in the early specification of the myeloid lineage.
Project description:Breast carcinoma amplified sequence 2 (BCAS2), a core component of the hPrP19 complex, plays crucial roles in various physiological and pathological processes. However, whether BCAS2 has functions other than being a key RNA-splicing regulator within the nucleus remains unknown. Here, we show that BCAS2 is essential for primitive hematopoiesis in zebrafish and mouse embryos. The activation of Wnt/β-catenin signal, which is required for hematopoietic progenitor differentiation, is significantly decreased upon depletion of bcas2 in zebrafish embryos and mouse embryonic fibroblasts (MEFs). Interestingly, BCAS2 deficiency has no obvious impact on the splicing efficiency of β-catenin pre-mRNA, while significantly attenuating β-catenin nuclear accumulation. Moreover, we find that BCAS2 directly binds to β-catenin via its coiled-coil domains, thereby sequestering β-catenin within the nucleus. Thus, our results uncover a previously unknown function of BCAS2 in promoting Wnt signaling by enhancing β-catenin nuclear retention during primitive hematopoiesis.
Project description:Proper control of inflammatory responses is essential for embryonic development, but the underlying mechanism is poorly understood. Here, we show that under physiological conditions, inactivation of ISG15, an inflammation amplifier, is associated with the interaction of Beclin 1 (Becn1), via its ECD domain, with STAT3 in the major fetal hematopoietic organ of mice. Conditional loss of Becn1 caused sequential dysfunction and exhaustion of fetal liver hematopoietic stem cells, leading to lethal inflammatory cell-biased hematopoiesis in the fetus. Molecularly, the absence of Becn1 resulted in the release of STAT3 from Becn1 tethering and subsequent phosphorylation and translocation to the nucleus, which in turn directly activated the transcription of ISG15 in fetal liver hematopoietic cells, coupled with increased ISGylation and production of inflammatory cytokines, whereas inactivating STAT3 reduced ISG15 transcription and inflammation but improved hematopoiesis potential, and further silencing ISG15 mitigated the above collapse in the Becn1 null hematopoietic lineage. The Becn1-STAT3-ISG15 axis remains functional in Atg5/7-disrupted fetal hematopoietic organs. These results suggest that Becn1, in an autophagy-independent manner, secures hematopoiesis and survival of the fetus by directly inhibiting STAT3-ISG15 activation to prevent cytokine storms. Our findings highlight a previously undocumented role of Becn1 in governing ISG15 to safeguard the fetus.
Project description:Developmental remodeling shapes neural circuits via activity-dependent pruning of synapses and axons. The cytoskeleton is critical for this process, as microtubule loss via enzymatic severing is an early step of pruning across many circuits and species. However, how microtubule-severing enzymes, such as spastin, are activated in specific neuronal compartments remains unknown. Here, we reveal that polyglutamylation, a posttranslational tubulin modification that is enriched in neurons, plays an instructive role in developmental remodeling by tagging microtubules for severing. Motor neuron-specific gene deletion of enzymes that add or remove tubulin polyglutamylation—TTLL glutamylases vs. CCP deglutamylases—accelerates or delays neuromuscular synapse remodeling in a neurotransmission-dependent manner. This mechanism is not specific to peripheral synapses but also operates in central circuits, e.g., the hippocampus. Thus, tubulin polyglutamylation acts as an activity-dependent rheostat of remodeling and shapes neuronal morphology and connectivity.
Project description:IKKa, a major regulator of noncanonical and canonical NF-kB pathways, is essential for B-lymphocyte maturation and secondary lymph organ formation. No evidence of IKKa regulating early B cell development currently exists. Here we found reduced pre-pro-B and pro-B cells but increased myeloid-erythroid lineages in the bone marrow (BM) of knockin mice expressing reduced and kinase-dead IKKa (KA/KA). The KA/KA BM cells recaptured their defects in wild-type recipients and KA/KA fetal liver displayed reduced B cells but increased progenitors. IKKa inactivation impaired both NF-kB pathways and deregulated expression of many genes required for early B cell commitment and hematopoiesis, including downregulated Pax5, IRF4, and Ikaros expression, but increased C/EBPa, GATA1, and Stat3 levels. Reintroduced combined NF-kB components, Pax5, and IKKa promoted BM B cell differentiation and repressed myeloid-erythroid lineages. Our studies revealed a new function of IKKa in a coordinated development process of B-lineage and erythroid-myeloid lineages during hematopoiesis via multiple pathways. Microarray analysis was performed on RNA isolated from the BM of B220+ cells isolated from 4-week old WT and KA/KA mice using affymetrix mouse 430 2.0 array chip, containing 45,000 genes, at the Laboratory of Molecular Technology SAIC-Frederick. Data were normalized, and log2 transformations were generated using Partek software (St. Louis, MO, USA).