Tuning apicobasal polarity and junctional recycling in the hemogenic endothelium orchestrates the morphodynamic complexity of emerging pre-hematopoietic stem cells.

Hematopoietic stem cells emerge in the embryo from an aortic-derived tissue called the hemogenic endothelium (HE). The HE appears to give birth to cells of different nature and fate but the molecular principles underlying this complexity are largely unknown. Here we show, in the zebrafish embryo, that two cell types emerge from the aortic floor with radically different morphodynamics. With the support of live imaging, we bring evidence suggesting that the mechanics underlying the two emergence types rely, or not, on apicobasal polarity establishment. While the first type is characterized by reinforcement of apicobasal polarity and maintenance of the apical/luminal membrane until release, the second type emerges via a dynamic process reminiscent of trans-endothelial migration. Interfering with Runx1 function suggests that the balance between the two emergence types depends on tuning apicobasal polarity at the level of the HE. In support of this and unexpectedly, we show that Pard3ba - one of the four Pard3 proteins expressed in the zebrafish - is sensitive to interference with Runx1 activity, in aortic endothelial cells. This supports the idea of a signaling cross talk controlling cell polarity and its associated features, between aortic and hemogenic cells. In addition, using new transgenic fish lines that express Junctional Adhesion Molecules and functional interference, we bring evidence for the essential role of ArhGEF11/PDZ-RhoGEF in controlling the HE-endothelial cell dynamic interface, including cell-cell intercalation, which is ultimately required for emergence completion. Overall, we highlight critical cellular and dynamic events of the endothelial-to-hematopoietic transition that support emergence complexity, with a potential impact on cell fate.

In mammals and other animals with backbones, the cells that will make up blood and immune cells are generated during a very narrow timeframe in embryonic development. These cells, called hematopoietic stem cells and progenitors (or HSPCs for short), emerge from tissue known as hemogenic endothelium that makes up the floor of early blood vessels. For HPSCs to eventually specialise into different types of blood and immune cells, they require diverse migratory and homing properties that, ultimately, will determine the specific type of functions they exert. An important question for scientists studying the development of different blood and immune cell types is when this commitment to functional diversity is established. It could, for example, arise due to cells in the hemogenic endothelium having different origins. Alternatively, the signals that generate hemogenic endothelium cells could be responsible. It is also possible that both explanations are true, and that having different mechanisms involved ensures diversity in populations of HSPCs. To investigate differences between the HSPCs emerging from the hemogenic endothelium, Torcq et al. studied zebrafish embryos that had been modified so that one of the proteins involved in sensing cell polarity ­ where the top and bottom of the cell are located ­ was fluorescent. Live imaging of the embryos showed that two types of cells, with striking differences in morphology, emerge from the hemogenic tissue. In addition, one cell type displays the same polarity as the other vessel cells, whereas the other does not. Torcq et al. also present evidence suggesting that the signals responsible for controlling this cell polarity are provided by surrounding blood vessel cells, supporting the idea of an interplay between the different cell types. The finding that two different cell types emerge from the hemogenic endothelium, reveals a potential new source of diversity in HSPCs. Ultimately, this is expected to contribute to their functional complexity, resulting in both long-term stem cells that retain their full regenerative potential into adulthood and more specialized blood and immune cells.

Deficiency of P2RY11 causes narcolepsy and attenuates the recruitment of neutrophils and macrophages in the inflammatory response in zebrafish.

Purinergic receptor P2Y11, a G protein-coupled receptor that is stimulated by extracellular ATP, has been demonstrated to be related to the chemotaxis of granulocytes, apoptosis of neutrophils, and secretion of cytokines in vitro. P2Y11 mutations were associated with narcolepsy. However, little is known about the roles of P2RY11 in the occurrence of narcolepsy and inflammatory response in vivo. In this study, we generated a zebrafish P2Y11 mutant using CRISPR/Cas9 genome editing and demonstrated that the P2Y11 mutant replicated the narcolepsy-like features including reduced HCRT expression and excessive daytime sleepiness, suggesting that P2Y11 is essential for HCRT expression. Furthermore, we accessed the cytokine expression in the mutant and revealed that the P2RY11 mutation disrupted the systemic inflammatory balance by reducing il4, il10 and tgfb, and increasing il6, tnfa, and il1b. In addition, the P2RY11-deficient larvae with caudal fin injuries exhibited significantly slower migration and less recruitment of neutrophils and macrophages at damaged site, and lower expression of anti-inflammatory cytokines during tissue damage. All these findings highlight the vital roles of P2RY11 in maintaining HCRT production and secreting anti-inflammatory cytokines in the native environment, and suggested that P2RY11-deficient zebrafish can serve as a reliable and unique model to further explore narcolepsy and inflammatory-related diseases with impaired neutrophil and macrophage responses.

ADA2 regulates inflammation and hematopoietic stem cell emergence via the A2bR pathway in zebrafish.

Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity caused by loss-of-function mutations in the adenosine deaminase 2 (ADA2) gene. Clinical manifestations of DADA2 include vasculopathy and immuno-hematological abnormalities, culminating in bone marrow failure. A major gap exists in our knowledge of the regulatory functions of ADA2 during inflammation and hematopoiesis, mainly due to the absence of an ADA2 orthologue in rodents. Exploring these mechanisms is essential for understanding disease pathology and developing new treatments. Zebrafish possess two ADA2 orthologues, cecr1a and cecr1b, with the latter showing functional conservation with human ADA2. We establish a cecr1b-loss-of-function zebrafish model that recapitulates the immuno-hematological and vascular manifestations observed in humans. Loss of Cecr1b disrupts hematopoietic stem cell specification, resulting in defective hematopoiesis. This defect is caused by induced inflammation in the vascular endothelium. Blocking inflammation, pharmacological modulation of the A2r pathway, or the administration of the recombinant human ADA2 corrects these defects, providing insights into the mechanistic link between ADA2 deficiency, inflammation and immuno-hematological abnormalities. Our findings open up potential therapeutic avenues for DADA2 patients.

Deciphering the function of the fifth class of Gα proteins: regulation of ionic homeostasis as unifying hypothesis.

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins.

Triptolide, a Cancer Cell Proliferation Inhibitor, Causes Zebrafish Muscle Defects by Regulating Notch and STAT3 Signaling Pathways.

Triptolide is a natural compound in herbal remedies with anti-inflammatory and anti-proliferative properties. We studied its effects on critical signaling processes within the cell, including Notch1 and STAT3 signaling. Our research showed that triptolide reduces cancer cell proliferation by decreasing the expression of downstream targets of these signals. The levels of each signal-related protein and mRNA were analyzed using Western blot and qPCR methods. Interestingly, inhibiting one signal with a single inhibitor alone did not significantly reduce cancer cell proliferation. Instead, MTT assays showed that the simultaneous inhibition of Notch1 and STAT3 signaling reduced cell proliferation. The effect of triptolide was similar to a combination treatment with inhibitors for both signals. When we conducted a study on the impact of triptolide on zebrafish larvae, we found that it inhibited muscle development and interfered with muscle cell proliferation, as evidenced by differences in the staining of myosin heavy chain and F-actin proteins in confocal fluorescence microscopy. Additionally, we noticed that inhibiting a single type of signaling did not lead to any significant muscle defects. This implies that triptolide obstructs multiple signals simultaneously, including Notch1 and STAT3, during muscle development. Chemotherapy is commonly used to treat cancer, but it may cause muscle loss due to drug-related adverse reactions or other complex mechanisms. Our study suggests that anticancer agents like triptolide, inhibiting essential signaling pathways including Notch1 and STAT3 signaling, may cause muscle atrophy through anti-proliferative activity.

Developmental Delay and Male-Biased Sex Ratio in esr2b Knockout Zebrafish.

The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Macrophage transplantation rescues RNASET2-deficient leukodystrophy by replacing deficient microglia in a zebrafish model.

RNASET2-deficient leukodystrophy is a rare infantile white matter disorder mimicking a viral infection and resulting in severe psychomotor impairments. Despite its severity, there is little understanding of cellular mechanisms of pathogenesis and no treatments. Recent research using the rnaset2 mutant zebrafish model has suggested that microglia may be the drivers of the neuropathology, due to their failure to digest apoptotic debris during neurodevelopment. Therefore, we developed a strategy for microglial replacement through transplantation of adult whole kidney marrow-derived macrophages into embryonic hosts. Using live imaging, we revealed that transplant-derived macrophages can engraft within host brains and express microglia-specific markers, suggesting the adoption of a microglial phenotype. Tissue-clearing strategies revealed the persistence of transplanted cells in host brains beyond embryonic stages. We demonstrated that transplanted cells clear apoptotic cells within the brain, as well as rescue overactivation of the antiviral response otherwise seen in mutant larvae. RNA sequencing at the point of peak transplant-derived cell engraftment confirms that transplantation can reduce the brain-wide immune response and particularly, the antiviral response, in rnaset2-deficient brains. Crucially, this reduction in neuroinflammation resulted in behavioral rescue-restoring rnaset2 mutant motor activity to wild-type (WT) levels in embryonic and juvenile stages. Together, these findings demonstrate the role of microglia as the cellular drivers of neuropathology in rnaset2 mutants and that macrophage transplantation is a viable strategy for microglial replacement in the zebrafish. Therefore, microglia-targeted interventions may have therapeutic benefits in RNASET2-deficient leukodystrophy.

A zebrafish model of diabetic nephropathy shows hyperglycemia, proteinuria and activation of the PI3K/Akt pathway.

Diabetic nephropathy (DN), as a complication of diabetes, is a substantial healthcare challenge owing to the high risk of morbidity and mortality involved. Although significant progress has been made in understanding the pathogenesis of DN, more efficient models are required to develop new therapeutics. Here, we created a DN model in zebrafish by crossing diabetic Tg(acta1:dnIGF1R-EGFP) and proteinuria-tracing Tg(l-fabp::VDBP-GFP) lines, named zMIR/VDBP. Overfed adult zMIR/VDBP fish developed severe hyperglycemia and proteinuria, which were not observed in wild-type zebrafish. Renal histopathology revealed human DN-like characteristics, such as glomerular basement membrane thickening, foot process effacement and glomerular sclerosis. Glomerular dysfunction was restored upon calorie restriction. RNA sequencing analysis demonstrated that DN zebrafish kidneys exhibited transcriptional patterns similar to those seen in human DN pathogenesis. Notably, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was activated, a phenomenon observed in the early phase of human DN. In addition, metformin improved hyperglycemia and proteinuria in DN zebrafish by modulating Akt phosphorylation. Our results indicate that zMIR/VDBP fish are suitable for elucidating the mechanisms underlying human DN and could be a powerful tool for therapeutic discovery.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Zebrafish nampt-a mutants are viable despite perturbed primitive hematopoiesis.

BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD+ in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been shown to cause abnormal development and defective hematopoiesis concomitant with decreased NAD+ levels. However, surprisingly, nampt-a mutant zebrafish were recently found to be viable, suggesting a discrepancy between the phenotypes in knockdown and knockout conditions. Here, we address this discrepancy by directly comparing loss-of-function approaches that result in identical defective transcripts in morphants and mutants. RESULTS: Using CRISPR/Cas9-mediated mutagenesis, we generated nampt-a mutant lines that carry the same mis-spliced mRNA as nampt-a morphants. Despite reduced NAD+ levels and perturbed expression of specific blood markers, nampt-a mutants did not display obvious developmental defects and were found to be viable. In contrast, injection of nampt-a morpholinos into wild-type or mutant nampt-a embryos caused aberrant phenotypes. Moreover, nampt-a morpholinos caused additional reduction of blood-related markers in nampt-a mutants, suggesting that the defects observed in nampt-a morphants can be partially attributed to off-target effects of the morpholinos. CONCLUSIONS: Our findings show that zebrafish nampt-a mutants are viable despite reduced NAD+ levels and a perturbed hematopoietic gene expression program, indicating strong robustness of primitive hematopoiesis during early embryogenesis.

A brain-specific angiogenic mechanism enabled by tip cell specialization.

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

The interaction of Pu.1 and cMyb in zebrafish neutrophil development.

Granulopoiesis is a highly ordered and precisely regulated process in which hematopoietic-related transcription factors play crucial roles. These transcription factors form complex regulatory networks through interactions with their co-factors or with each other, and anomalies in these networks can lead to the onset of leukemia. While the structures and functions of dozens of transcription factors involved in this process have been extensively studied, research on the regulatory relationships between these factors remains relatively limited. PU.1 and cMYB participate in multiple stages of neutrophil development, and their abnormalities are often associated with hematologic disorders. However, the regulatory relationship between these factors in vivo and their mode of interaction remain unclear. In this study, zebrafish models with cMyb overexpression (cmybhyper) and Pu.1 deficiency (pu.1G242D/G242D) were utilized to systematically investigate the interaction between Pu.1 and cMyb during granulopoiesis through whole-mount in situ hybridization, qRT-PCR, fluorescence reporting systems, and rescue experiments. The results showed a significant increase in cmyb expression in neutrophils of the pu.1G242D/G242D mutant, while there was no apparent change in pu.1 expression in cmybhyper. Further experiments involving injection of morpholino (MO) to decrease cmyb expression in pu.1G242D/G242D mutants, followed by SB and BrdU staining to assess neutrophil quantity and proliferation, revealed that reducing cmyb expression could rescue the abnormal proliferation phenotype of neutrophils in the pu.1G242D/G242D mutant. These findings suggest that Pu.1 negatively regulates the expression of cMyb during neutrophil development. Finally, through the construction of multi-site mutation plasmids and a fluorescent reporter system, confirmed that Pu.1 directly binds to the +72 bp site in the cmyb promoter, exerting negative regulation on its expression. In conclusion, this study delineates that Pu.1 participates in neutrophil development by regulating cmyb expression. This provides new insights into the regulatory relationship between these two factors and their roles in diseases.

Identification of Dhx15 as a Major Regulator of Liver Development, Regeneration, and Tumor Growth in Zebrafish and Mice.

RNA helicase DHX15 plays a significant role in vasculature development and lung metastasis in vertebrates. In addition, several studies have demonstrated the overexpression of DHX15 in the context of hepatocellular carcinoma. Therefore, we hypothesized that this helicase may play a significant role in liver regeneration, physiology, and pathology. Dhx15 gene deficiency was generated by CRISPR/Cas9 in zebrafish and by TALEN-RNA in mice. AUM Antisense-Oligonucleotides were used to silence Dhx15 in wild-type mice. The hepatocellular carcinoma tumor induction model was generated by subcutaneous injection of Hepa 1-6 cells. Homozygous Dhx15 gene deficiency was lethal in zebrafish and mouse embryos. Dhx15 gene deficiency impaired liver organogenesis in zebrafish embryos and liver regeneration after partial hepatectomy in mice. Also, heterozygous mice presented decreased number and size of liver metastasis after Hepa 1-6 cells injection compared to wild-type mice. Dhx15 gene silencing with AUM Antisense-Oligonucleotides in wild-type mice resulted in 80% reduced expression in the liver and a significant reduction in other major organs. In addition, Dhx15 gene silencing significantly hindered primary tumor growth in the hepatocellular carcinoma experimental model. Regarding the potential use of DHX15 as a diagnostic marker for liver disease, patients with hepatocellular carcinoma showed increased levels of DHX15 in blood samples compared with subjects without hepatic affectation. In conclusion, Dhx15 is a key regulator of liver physiology and organogenesis, is increased in the blood of cirrhotic and hepatocellular carcinoma patients, and plays a key role in controlling hepatocellular carcinoma tumor growth and expansion in experimental models.

EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes ß-catenin signaling during hematopoietic stem cell development in zebrafish.

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

TAF1 is needed for the proliferation and maturation of thyroid follicle cells via Notch signaling.

Thyroid dysgenesis (TD) is the common pathogenic mechanism of congenital hypothyroidism (CH). In addition, known pathogenic genes are limited to those that are directly involved in thyroid development. To identify additional candidate pathogenetic genes, we performed forward genetic screening for TD in zebrafish, followed by positional cloning. The candidate gene was confirmed in vitro using the Nthy-ori 3.1 cell line and in vivo using a zebrafish model organism. We obtained a novel zebrafish line with thyroid dysgenesis and identified the candidate pathogenetic mutation TATA-box binding protein associated Factor 1 (taf1) by positional cloning. Further molecular studies revealed that taf1 was needed for the proliferation of thyroid follicular cells by binding to the NOTCH1 promoter region. Knockdown of TAF1 impaired the proliferation and maturation of thyroid cells, thereby leading to thyroid dysplasia. This study showed that TAF1 promoted Notch signaling and that this association played a pivotal role in thyroid development.NEW & NOTEWORTHY In our study, we obtained a novel zebrafish line with thyroid dysgenesis (TD) and identified the candidate pathogenetic mutation TATA-box binding protein associated Factor 1 (taf1). Further researches revealed that taf1 was required for thyroid follicular cells by binding to the NOTCH1 promoter region. Our findings revealed a novel role of TAF1 in thyroid morphogenesis.

ptx3a+ fibroblast/epicardial cells provide a transient macrophage niche to promote heart regeneration.

Macrophages conduct critical roles in heart repair, but the niche required to nurture and anchor them is poorly studied. Here, we investigated the macrophage niche in the regenerating heart. We analyzed cell-cell interactions through published single-cell RNA sequencing datasets and identified a strong interaction between fibroblast/epicardial (Fb/Epi) cells and macrophages. We further visualized the association of macrophages with Fb/Epi cells and the blockage of macrophage response without Fb/Epi cells in the regenerating zebrafish heart. Moreover, we found that ptx3a+ epicardial cells associate with reparative macrophages, and their depletion resulted in fewer reparative macrophages. Further, we identified csf1a expression in ptx3a+ cells and determined that pharmacological inhibition of the csf1a pathway or csf1a knockout blocked the reparative macrophage response. Moreover, we found that genetic overexpression of csf1a enhanced the reparative macrophage response with or without heart injury. Altogether, our studies illuminate a cardiac Fb/Epi niche, which mediates a beneficial macrophage response after heart injury.

Stat3 Regulates Developmental Hematopoiesis and Impacts Myeloid Cell Function via Canonical and Non-Canonical Modalities.

INTRODUCTION: Signal transducer and activator of transcription (STAT) 3 is extensively involved in the development, homeostasis, and function of immune cells, with STAT3 disruption associated with human immune-related disorders. The roles ascribed to STAT3 have been assumed to be due to its canonical mode of action as an inducible transcription factor downstream of multiple cytokines, although alternative noncanonical functional modalities have also been identified. The relative involvement of each mode was further explored in relevant zebrafish models. METHODS: Genome editing with CRISPR/Cas9 was used to generate mutants of the conserved zebrafish Stat3 protein: a loss of function knockout (KO) mutant and a mutant lacking C-terminal sequences including the transactivation domain (ΔTAD). Lines harboring these mutations were analyzed with respect to blood and immune cell development and function in comparison to wild-type zebrafish. RESULTS: The Stat3 KO mutant showed perturbation of hematopoietic lineages throughout primitive and early definitive hematopoiesis. Neutrophil numbers did not increase in response to lipopolysaccharide (LPS) or granulocyte colony-stimulating factor (G-CSF) and their migration was significantly diminished, the latter correlating with abrogation of the Cxcl8b/Cxcr2 pathway, with macrophage responses perturbed. Intriguingly, many of these phenotypes were not shared by the Stat3 ΔTAD mutant. Indeed, only neutrophil and macrophage development were disrupted in these mutants with responsiveness to LPS and G-CSF maintained, and neutrophil migration actually increased. CONCLUSION: This study has identified roles for zebrafish Stat3 within hematopoietic stem cells impacting multiple lineages throughout primitive and early definitive hematopoiesis, myeloid cell responses to G-CSF and LPS and neutrophil migration. Many of these roles showed conservation, but notably several involved noncanonical modalities, providing additional insights for relevant diseases.

A Mettl16/m6A/mybl2b/Igf2bp1 axis ensures cell cycle progression of embryonic hematopoietic stem and progenitor cells.

Prenatal lethality associated with mouse knockout of Mettl16, a recently identified RNA N6-methyladenosine (m6A) methyltransferase, has hampered characterization of the essential role of METTL16-mediated RNA m6A modification in early embryonic development. Here, using cross-species single-cell RNA sequencing analysis, we found that during early embryonic development, METTL16 is more highly expressed in vertebrate hematopoietic stem and progenitor cells (HSPCs) than other methyltransferases. In Mettl16-deficient zebrafish, proliferation capacity of embryonic HSPCs is compromised due to G1/S cell cycle arrest, an effect whose rescue requires Mettl16 with intact methyltransferase activity. We further identify the cell-cycle transcription factor mybl2b as a directly regulated by Mettl16-mediated m6A modification. Mettl16 deficiency resulted in the destabilization of mybl2b mRNA, likely due to lost binding by the m6A reader Igf2bp1 in vivo. Moreover, we found that the METTL16-m6A-MYBL2-IGF2BP1 axis controlling G1/S progression is conserved in humans. Collectively, our findings elucidate the critical function of METTL16-mediated m6A modification in HSPC cell cycle progression during early embryonic development.

Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

BACKGROUND: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. METHODS: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. RESULTS: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. CONCLUSIONS: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.