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Hematopoietic stem cells (HSCs) are rare cells that arise in the embryo and sustain adult hematopoiesis. Although the functional potential of nascent HSCs is detectable by transplantation, their native contribution during development is unknown, in part due to the overlapping genesis and marker gene expression with other embryonic blood progenitors. Using single-cell transcriptomics, we define gene signatures that distinguish nascent HSCs from embryonic blood progenitors. Applying a lineage-tracing approach to selectively track HSC output in situ, we find significantly delayed lymphomyeloid contribution. An inducible HSC injury model demonstrates a negligible impact on larval lymphomyelopoiesis following HSC depletion. HSCs are not merely dormant at this developmental stage, as they showed robust regeneration after injury. Combined, our findings illuminate that nascent HSCs self-renew but display differentiation latency, while HSC-independent embryonic progenitors sustain developmental hematopoiesis. Understanding these differences could improve de novo generation and expansion of functional HSCs.

Valosin-containing protein (VCP) mutations cause inclusion body myopathy with Paget disease and frontotemporal dementia. However, the mechanisms by which mutant VCP triggers degeneration remain unknown. Here, we investigated the role of VCP in cellular stress and found that the oxidative stressor arsenite and heat shockdactivated stress responses evident by T-intracellular antigen-1epositive granules in C2C12 myoblasts. Granules also contained phosphorylated transactive response DNA-binding protein 43, ubiquitin, microtubule-associated protein 1A/1B light chains 3, and lysosome-associated membrane protein 2. Mutant VCP produced more T-intracellular antigen-1epositive granules than wildtype in the postarsenite exposure period. Similar results were observed for other granule components, indicating that mutant VCP delayed clearance of stress granules. Furthermore, stress granule resolution was impaired on differentiated C2C12 cells expressing mutant VCP. To address whether mutant VCP triggers dysregulation of the stress granule pathway in vivo, we analyzed skeletal muscle of aged VCP R155H -knockin mice. We found significant increments in oxidated proteins but observed the stress granule markers RasGAP SH3-binding protein and phosphorylated eukaryotic translation initiation factor 2a unchanged. The mixed results indicate that mutant VCP together with aging lead to higher oxidative stress in skeletal muscle but were insufficient to disrupt the stress granule pathway. Our findings support that deficiencies in recovery from stressors may result in attenuated tolerance to stress that could trigger muscle degeneration. (Am J Pathol 2016 http://dx

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Ten eleven translocation 1 (Tet1) is a DNA dioxygenase that promotes DNA demethylation by oxidizing 5-methylcytosine. It can also partner with chromatin-activating and repressive complexes to regulate gene expressions independent of its enzymatic activity. Tet1 is highly expressed in embryonic stem cells (ESCs) and regulates pluripotency and differentiation. However, its roles in ESC cell cycle progression and proliferation have not been investigated. Using a series of Tet1 catalytic mutant (Tet1m/m), knockout (Tet1−/−) and wild type (Tet1+/+) mouse ESCs (mESCs), we identified a non-catalytic role of Tet1 in the proper cell cycle progression and proliferation of mESCs. Tet1−/−, but not Tet1m/m, mESCs exhibited a significant reduction in proliferation and delayed progression through G1. We found that the cyclin-dependent kinase inhibitor p21/Cdkn1a was uniquely upregulated in Tet1−/− mESCs and its knockdown corrected the slow proliferation and delayed G1 progression. Mechanistically, we found that p21 was a direct target of Tet1. Tet1 occupancy at the p21 promoter overlapped with the repressive histone mark H3K27me3 as well as with the H3K27 trimethyl transferase PRC2 component Ezh2. A loss of Tet1, but not loss of its catalytic activity, significantly reduced the enrichment of Ezh2 and H3K27 trimethylation at the p21 promoter without affecting the DNA methylation levels. We also found that the proliferation defects of Tet1−/− mESCs were independent of their differentiation defects. Together, these findings established a non-catalytic role for Tet1 in suppressing p21 in mESCs to ensure a rapid G1-to-S progression, which is a key hallmark of ESC proliferation. It also established Tet1 as an epigenetic regulator of ESC proliferation in addition to its previously defined roles in ESC pluripotency and differentiation.

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Definitive hematopoietic stem cells (HSCs) emerge in the embryo and sustain major adult hematopoietic lineages. Although their functional potential is detected by transplant, nascent HSC contribution during development is both unknown and difficult to address due to the overlapping emergence of HSC-independent progenitors-cells that lack the multipotency and/or longevity of HSCs but express many of the same markers. Using sorted hematopoietic stem and progenitor cells from zebrafish embryos, we performed single cell RNA sequencing to decipher HSC and HSC-independent progenitor heterogeneity during the time frame of their emergence and initial maturation. After batch correction and dimensional reduction, we identified seven distinct populations that are inferred from RNA velocity analysis to originate from pre-hemogenic endothelium and develop into three main differentiation trajectories. We also determined that HSCs can be distinguished from HSC-independent progenitors based on the temporal regulation and differential activity of the draculin (drl) promoter that was previously shown to mark adult-contributing HSCs. From these studies, we found that the drl promoter is active in HSCs and HSC-independent progenitors at 1-day post-fertilization (dpf) but becomes highly expressed primarily in HSCs by 2 dpf. We applied a drl:cre-ER T2 tamoxifen-inducible Cre-loxP lineage-tracing approach to selectively lineage trace HSCs starting at 2 dpf and track their myeloid and lymphoid contribution during larval development and adulthood. We determined that HSC-independent progenitors primarily contribute to developmental lymphomyelopoiesis with minimal HSC contribution until after 7 dpf. Consistent with this result, we demonstrated that although HSCs robustly regenerated after hematopoietic injury using a novel inducible larval HSC injury model, their depletion had almost no impact on lymphoid and myeloid cell numbers up to 7 dpf. These findings suggest that HSCs are not entirely dormant during development and that there exists an uncoupling of HSC self-renewal and differentiation in development. In conclusion, we determine that it is the HSC-independent progenitors, and not HSCs, that sustain embryonic and early larval lymphomyelopoiesis. Acquiring a greater understanding regarding developmental differences in progenitor and HSC specification and maturation will inform and improve the generation of functional HSCs from renewable pluripotent stem cells. Disclosures No relevant conflicts of interest to declare.

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