MLL-AF9 regulates transcriptional initiation in mixed lineage leukemic cells.

MLL-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce mixed lineage leukemia (MLL) through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II (Pol II) in HEL, a human cell line without MLL1 arrangement (MLLr). MLL1 and AF9 only co-regulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and MLL-AF9 occupy promoters and distal intergenic regions, exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia (AML) cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely-believed elongation.

AFF4 globally affects the release of paused RNA polymerase II in HEL cells.

P-TEFb, a heterodimer of the kinase CDK9 and Cyclin T1, is a critical regulator of promoter-proximal pause release of Pol II in metazoans. It is capable of forming three larger complexes, including the super elongation complex (SEC), the BRD4/P-TEFb complex and the 7SK snRNP. In the SEC or the BRD4/P-TEFb complex, P-TEFb is enzymatically active, while in the 7SK snRNP, its activity is inhibited. The SEC consists of AFF1 or 4, ENL or AF9, ELL1, 2 or 3 and EAF1 or 2 in addition to P-TEFb, the only subunit with catalytic activity, and the noncatalytic subunits have been found to be able to regulate pause release through P-TEFb. We and others recently found that AFF1, ENL and AF9 are capable of regulating transcriptional initiation, but it is unknown yet whether AFF4 is also capable of doing so. With respect to the gene regulation selectivity of the SEC and the BRD4/P-TEFb complex, one recent study showed that in human DLD-1 cells, the SEC only regulates pause release of heat shock (HS) genes, whereas the BRD4/P-TEFb complex regulates pause release of the rest of the genes. However, it is unclear whether those mechanisms are general. In this study for the purpose of further understanding the role of AFF4 in transcriptional regulation, we found that AFF4 knockdown by RNA interference in human HEL cells decreased not only cellular level but also global chromatin occupancy of CTD serine 2 phosphorylated Pol II. Direct target genes of AFF4 were identified by RNA-seq and CUT&Tag. Notably, we found by ChIP-seq and PRO-seq that AFF4 loss also increased promoter-proximal pause of Pol II on several hundred HS and thousands of non-HS genes. Mechanistically, AFF4 promotes pause release likely by facilitating the binding of P-TEFb to Pol II. These results suggest that extent of the impact of AFF4 on pause release is likely to be context-dependent or cell-type dependent.

A systematic comparison of anti-angiogenesis efficacy and cardiotoxicity of receptor tyrosine kinase inhibitors in zebrafish model.

Pathological angiogenesis is fundamental to progression of cancerous tumors and blinding eye diseases. Anti-angiogenic receptor tyrosine kinase inhibitors (TKIs) are in broad use for the treatment of these diseases. With more and more TKIs available, it is a challenge to make an optimal choice. It remains unclear whether TKIs demonstrate similar anti-angiogenesis activities in different tissues. Many TKIs have shown varying degrees of toxic effects that should also be considered in clinical use. This study investigates the anti-angiogenic effects of 13 FDA-approved TKIs on the intersegmental vessels (ISVs), subintestinal vessels (SIVs) and retinal vasculature in zebrafish embryos. The results show that vascular endothelial growth factor receptor TKIs (VEGFR-TKIs) exhibit anti-angiogenic abilities similarly on ISVs and SIVs, and their efficacy is consistent with their IC50 values against VEGFR2. In addition, VEGFR-TKIs selectively induces the apoptosis of endothelial cells in immature vessels. Among all TKIs tested, axitinib demonstrates a strong inhibition on retinal neovascularization at a low dose that do not strongly affect ISVs and SIVs, supporting its potential application for retinal diseases. Zebrafish embryos demonstrate cardiotoxicity after VEGFR-TKIs treatment, and ponatinib and sorafenib show a narrow therapeutic window, suggesting that these two drugs may need to be dosed more carefully in patients. We propose that zebrafish is an ideal model for studying in vivo antiangiogenic efficacy and cardiotoxicity of TKIs.

Zebrafish Xenograft Model for Studying Pancreatic Cancer-Instructed Innate Immune Microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) has up to half the tumor mass of tumor-associated myeloid cells. Myeloid innate immune cells play important roles in regulating cancer cell recognition and tumor growth. PDAC cells often mold myeloid cells into pro-tumoral state to fuel cancer growth and induce immune suppression. However, how tumor cells educate the innate immune responses remains largely unknown. In this study, we used four different human PDAC cell lines (PANC1, BxPC3, AsPC1, and CFPAC1) to establish the zebrafish xenograft model and investigated the interaction between pancreatic cancer and innate immune cells. The primary tumor-derived cancer cells PANC1 and BxPC3 activated innate immune anti-tumoral responses efficiently, while cancer cells from metastatic tissues AsPC1 and CFPAC1 induced an innate immune suppression and educated innate immune cells towards pro-tumoral state. Chemical conversion of innate immune cells to anti-tumoral state inhibited tumor growth for AsPC1 and CFPAC1. Moreover, genetic and pharmacological inhibition of macrophages also significantly reduced tumor growth, supporting the important roles of macrophages in innate immune suppression. REG4 expression is high in AsPC1 and CFPAC1. Knockdown of REG4 induced innate immune activation and reduced tumor growth in the xenografts, indicating that REG4 is a beneficial target for PDAC therapy. Our study provides a fast in-vivo model to study PDAC-innate immune interaction and their plasticity that could be used to study the related mechanism as well as identify new drugs to enhance immunotherapy.

Negative Elongation Factor (NELF) Inhibits Premature Granulocytic Development in Zebrafish.

Gene expression is tightly regulated during hematopoiesis. Recent studies have suggested that RNA polymerase II (Pol II) promoter proximal pausing, a temporary stalling downstream of the promoter region after initiation, plays a critical role in regulating the expression of various genes in metazoans. However, the function of proximal pausing in hematopoietic gene regulation remains largely unknown. The negative elongation factor (NELF) complex is a key factor important for this proximal pausing. Previous studies have suggested that NELF regulates granulocytic differentiation in vitro, but its in vivo function during hematopoiesis remains uncharacterized. Here, we generated the zebrafish mutant for one NELF complex subunit Nelfb using the CRISPR-Cas9 technology. We found that the loss of nelfb selectively induced excessive granulocytic development during primitive and definitive hematopoiesis. The loss of nelfb reduced hematopoietic progenitor cell formation and did not affect erythroid development. Moreover, the accelerated granulocytic differentiation and reduced progenitor cell development could be reversed by inhibiting Pol II elongation. Further experiments demonstrated that the other NELF complex subunits (Nelfa and Nelfe) played similar roles in controlling granulocytic development. Together, our studies suggested that NELF is critical in controlling the proper granulocytic development in vivo, and that promoter proximal pausing might help maintain the undifferentiated state of hematopoietic progenitor cells.

Dual-modal imaging with non-contact photoacoustic microscopy and fluorescence microscopy.

Simultaneous imaging of complementary absorption and fluorescence contrasts with high spatial resolution is useful for biomedical studies. However, conventional dual-modal photoacoustic (PA) and fluorescence imaging systems require the use of acoustic coupling media due to the contact operation of PA imaging, which causes issues and complicates the procedure in certain applications such as cell imaging and ophthalmic imaging. We present a novel dual-modal imaging system which combines non-contact PA microscopy (PAM) based on PA remote sensing and fluorescence microscopy (FLM) into one platform. The system enables high lateral resolution of 2 and 2.7 µm for PAM and FLM modes, respectively. In vivo imaging of a zebrafish larva injected with a rhodamine B solution is demonstrated, with PAM visualizing the pigment and FLM revealing the injected rhodamine B.

Miniature non-contact photoacoustic probe based on fiber-optic photoacoustic remote sensing microscopy.

Photoacoustic (PA) remote sensing (PARS) microscopy, featured by non-contact operation, has shown great potential for PA microscopy (PAM) imaging applications. However, current PARS microscopy systems are mainly based on free-space light, making the imaging head bulky and inconvenient to use. These issues hinder selected applications such as PA endoscopy and handheld PAM. Here, we report a miniature probe capable of non-contact PAM based on PARS microscopy. By utilizing fiber-optic components including a wavelength division multiplexer and an optical circulator, the imaging head can be highly miniaturized with a diameter of ∼3.0mm. Also, since all light is transmitted via fibers, the fiber-optic PARS microscopy system is relatively easy to build and facilitates scanning of the probe. In vivo imaging of a zebrafish larva and imaging of lithium metal batteries are conducted using the probe, showing its good imaging capability.

Photoacoustic-fluorescence microendoscopy in vivo.

A miniature endoscope capable of imaging multiple tissue contrasts in high resolution is highly attractive, because it can provide complementary and detailed tissue information of internal organs. Here we present a photoacoustic (PA)-fluorescence (FL) endoscope for optical-resolution PA microscopy (PAM) and FL microscopy (FLM). The endoscope with a diameter of 2.8 mm achieves high lateral resolutions of 5.5 and 6.3 µm for PAM and FLM modes, respectively. In vivo imaging of zebrafish larvae and a mouse ear is conducted, and high-quality images are obtained. Additionally, in vivo endoscopic imaging of a rat rectum is demonstrated, showing the endoscopic imaging capability of our endoscope. By providing dual contrasts with high resolution, the endoscope may open up new opportunities for clinical endoscopic imaging applications.

Laparoscopic S7 hepatectomy for hepatic mucinous neoplasm: a case report and literature review.

BACKGROUND: Mucinous cystic neoplasm of the Liver is rare tumors with malignant potential that occur in the biliary epithelium. Because of its rare presentation, it is often misdiagnosed before surgery. CASE PRESENTATION: A 63-year-old female patient presented with intermittent upper abdominal pain for three months. Laparoscopic hepatectomy of Segment 7 was conducted based on the preoperative diagnosis of space-occupying lesion in the right lobe of the liver. Postoperative pathology showed a low-grade mucinous cystic neoplasm in the right posterior lobe of the liver. The preoperative CA19-9 level was significantly increased while the postoperative CA19-9 returned to the normal range. CONCLUSIONS: The diagnosis of mucinous cystic neoplasm of the liver is closely related to the thickening of the cystic wall or the increase of CA19-9, which has great significance and deserves clinical attention.

A new aminoglycoside etimicin shows low nephrotoxicity and ototoxicity in zebrafish embryos.

Aminoglycoside antibiotics are widely used for many life-threatening infections. The use of aminoglycosides is often comprised by their deleterious side effects to the kidney and inner ear. A novel semisynthetic antibiotic, etimicin, has good antimicrobial activity against both gram-positive and gram-negative bacteria. But its toxicity profile analysis is still lacking. In the present study, we compared the in vivo toxic effects of three aminoglycosides, gentamicin, amikacin, and etimicin, in zebrafish embryos. We examined the embryotoxicity, nephrotoxicity, and the damage to the neuromast hair cells. Our results revealed that etimicin and amikacin exhibit more developmental toxicities to the young embryos than gentamicin. But at subtoxic doses, etimicin and amikacin show significantly reduced toxicities towards kidney and neuromast hair cells. We further demonstrated that fluorescently conjugated aminoglycosides (gentamicin-Texas red [GTTR], amikacin-Texas red [AMTR], and etimicin-Texas red [ETTR]) all enter the hair cells properly. Inside the hair cells, gentamicin, not etimicin and amikacin, displays robust reactive oxygen species generation and induces apoptosis. Our data support that the different intracellular cytotoxicity underlies the different ototoxicity of the three aminoglycosides and that etimicin is a new aminoglycoside with reduced risk of nephrotoxicity and ototoxicity.

Optomechanical Time-Gated Fluorescence Imaging Using Long-Lived Silicon Quantum Dot Nanoparticles.

We demonstrate a novel optomechanical synchronization method to achieve ultrahigh-contrast time-gated fluorescence imaging using live zebrafish as models. Silicon quantum dot nanoparticles (SiQDNPs) with photoluminescence lifetime of about 16 µs were used as the long-lived probes to enable background autofluorescence removal and multiplexing through time-gating. A continuous-wave 405 nm laser as the excitation source was focused on a rotating optical chopper on which the emission light beam obtained from an inverted fluorescence microscope was also focused but with a phase difference such that in a short delay after the excitation laser is blocked, the emission light beam passes through the optical chopper, initiating the image acquisition by a conventional sensor. Both excitation and detection time windows were synchronized by one optical chopper, eliminating the need for pulsed light source and image intensifier which is often used as ultrafast optical shutter. Through use of the cost-effective time-gating method, nearly all background autofluorescence emitted from the yolk sac of a zebrafish embryo microinjected with the SiQDNPs was removed, leading to a 45-fold increase in signal-to-background ratio. Furthermore, two kinds of fluorescence signals emitted from the microinjected SiQDNPs and the intrinsic green fluorescent protein of transgenic zebrafish larvae can be clearly separated through time-gating.

Mutations in QARS, encoding glutaminyl-tRNA synthetase, cause progressive microcephaly, cerebral-cerebellar atrophy, and intractable seizures.

Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.

Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4).

Recent small RNA sequencing data has uncovered 3' end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3' mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3' mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3' mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3' miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.

Astaxanthin inhibits apoptosis in alveolar epithelial cells type II in vivo and in vitro through the ROS-dependent mitochondrial signalling pathway.

Oxidative stress is an important molecular mechanism underlying lung fibrosis. The mitochondrion is a major organelle for oxidative stress in cells. Therefore, blocking the mitochondrial signalling pathway may be the best therapeutic manoeuver to ameliorate lung fibrosis. Astaxanthin (AST) is an excellent antioxidant, but no study has addressed the pathway of AST against pulmonary oxidative stress and free radicals by the mitochondrion-mediated signalling pathway. In this study, we investigated the antioxidative effects of AST against H2 O2 - or bleomycin (BLM)-induced mitochondrial dysfunction and reactive oxygen species (ROS) production in alveolar epithelial cells type II (AECs-II) in vivo and in vitro. Our data show that AST blocks H2 O2 - or BLM-induced ROS generation and dose-dependent apoptosis in AECs-II, as characterized by changes in cell and mitochondria morphology, translocation of apoptotic proteins, inhibition of cytochrome c (Cyt c) release, and the activation of caspase-9, caspase-3, Nrf-2 and other cytoprotective genes. These data suggest that AST inhibits apoptosis in AECs-II cells through the ROS-dependent mitochondrial signalling pathway and may be of potential therapeutic value in lung fibrosis treatment.

Analysing the relationship between lncRNA and protein-coding gene and the role of lncRNA as ceRNA in pulmonary fibrosis.

Long non-coding RNAs (lncRNAs) are involved in various pathophysiologic processes and human diseases. However, their dynamics and corresponding functions in pulmonary fibrosis remain poorly understood. In this study, portions of lncRNAs adjacent or homologous to protein-coding genes were determined by searching the UCSC genome bioinformatics database. This was found to be potentially useful for exploring lncRNA functions in disease progression. Previous studies showed that competing endogenous RNA (ceRNA) hypothesis is another method to predict lncRNA function. However, little is known about the function of ceRNA in pulmonary fibrosis. In this study, we selected two differentially expressed lncRNAs MRAK088388 and MRAK081523 to explore their regulatory mechanisms. MRAK088388 and MRAK081523 were analysed as long-intergenic non-coding RNAs (lincRNAs), and identified as orthologues of mouse lncRNAs AK088388 and AK081523, respectively. qRT-PCR and in situ hybridization (ISH) showed that they were significantly up-regulated, and located in the cytoplasm of interstitial lung cells. We also showed that MRAK088388 and N4bp2 had the same miRNA response elements (MREs) for miR-200, miR-429, miR-29, and miR-30, whereas MRAK081523 and Plxna4 had the same MREs for miR-218, miR-141, miR-98, and let-7. Moreover, the expression levels of N4bp2 and Plxna4 significantly increased in fibrotic rats, and were highly correlated with those of MRAK088388 and MRAK081523, respectively. Among their shared miRNAs, miR-29b-3p and let-7i-5p decreased in the model group, and were negatively correlated with the expression of MRAK088388 and MRAK081523, respectively. MRAK088388 and MRAK081523 could regulate N4bp2 and Plxna4 expression by sponging miR-29b-3p and let-7i-5p, respectively, and possessed regulatory functions as ceRNAs. Thus, our study may provide insights into the functional interactions of lncRNA, miRNA and mRNA, and lead to new theories for the pathogenesis and treatment of pulmonary fibrosis.

MiRNA-30a inhibits AECs-II apoptosis by blocking mitochondrial fission dependent on Drp-1.

Apoptosis of type II alveolar epithelial cells (AECs-II) is a key determinant of initiation and progression of lung fibrosis. However, the mechanism of miR-30a participation in the regulation of AECs-II apoptosis is ambiguous. In this study, we investigated whether miR-30a could block AECs-II apoptosis by repressing mitochondrial fission dependent on dynamin-related protein-1 (Drp-1). The levels of miR-30a in vivo and in vitro were determined through quantitative real-time PCR (qRT-PCR). The inhibition of miR-30a in AECs-II apoptosis, mitochondrial fission and its dependence on Drp-1, and Drp-1 expression and translocation were detected using miR-30a mimic, inhibitor-transfection method (gain- and loss-of-function), or Drp-1 siRNA technology. Results showed that miR-30a decreased in lung fibrosis. Gain- and loss-of-function studies revealed that the up-regulation of miR-30a could decrease AECs-II apoptosis, inhibit mitochondrial fission, and reduce Drp-1 expression and translocation. MiR-30a mimic/inhibitor and Drp-1 siRNA co-transfection showed that miR-30a could inhibit the mitochondrial fission dependent on Drp-1. This study demonstrated that miR-30a inhibited AECs-II apoptosis by repressing the mitochondrial fission dependent on Drp-1, and could function as a novel therapeutic target for lung fibrosis.

Multi-technology integrated network pharmacology-based study on phytochemicals, active metabolites, and molecular mechanism of Psoraleae Fructus to promote melanogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Compendium of Materia Medica (Shizhen Li, Ming dynasty) and Welfare Pharmacy (Song dynasty), Psoraleae Fructus (PF), a traditional Chinese medicine (TCM) has a bitter taste and warm nature, which has the effect of treating spleen and kidney deficiency and skin disease. Although PF has been widely used since ancient times and has shown satisfactory efficacy in treating vitiligo, the active substances and the mechanism of PF in promoting melanogenesis remain unclear. AIM OF THE STUDY: To explore the active substances and action mechanisms of PF in promoting melanogenesis. MATERIALS AND METHODS: Firstly, UPLC-UV-Q-TOF/MS was used to characterize the components in PF extract and identify the absorption components and metabolites of PF after oral administration at usual doses in rats. Secondly, the active substances and related targets and pathways were predicted by network pharmacology and molecular docking. Finally, pharmacodynamic and molecular biology experiments were used to verify the prediction results. RESULTS: The experimental results showed that 15 compounds were identified in PF extract, and 44 compounds, consisting of 8 prototype components and 36 metabolites (including isomers) were identified in rats' plasma. Promising action targets (MAPK1, MAPK8, MAPK14) and signaling pathways (MAPK signaling pathway) were screened and refined to elucidate the mechanism of PF against vitiligo based on network pharmacology. Bergaptol and xanthotol (the main metabolites of PF), psoralen (prototype drug), and PF extract significantly increased melanin production in zebrafish embryos. Furthermore, bergaptol could promote the pigmentation of zebrafish embryos more than psoralen and PF extract. Bergaptol significantly increased the protein expression levels of p-P38 and decreased ERK phosphorylation in B16F10 cells, which was also supported by the corresponding inhibitor/activator combination study. Moreover, bergaptol increased the mRNA expression levels of the downstream microphthalmia-associated transcription factor (MITF) and tyrosinase in B16F10 cells. Our data elucidate that bergaptol may promote melanogenesis by regulating the p-P38 and p-ERK signaling pathway. CONCLUSIONS: This study will lay a foundation for discovering potential new drugs for treating vitiligo and provide feasible ideas for exploring the mechanism of traditional Chinese medicine.

Assaying hematopoiesis using zebrafish.

The zebrafish has become a commonly used model for studying hematopoiesis as a result of its unique attributes. Zebrafish are highly suitable for large-scale genetic and chemical screens compared to other vertebrate systems. It is now possible to analyze hematopoietic lineages in zebrafish and validate cell function via transplantation assays. Here, we review advancements over the past decade in forward genetic screens, chemical screens, fluorescence-activated cell sorting analysis, and transplantation assays. Integrating these approaches enables new chemical and genetic screens that assay cell function within the hematopoietic system. Studies in zebrafish will continue to contribute and expand our knowledge about hematopoiesis, and develop novel treatments for clinical applications.

The microbiota regulates hematopoietic stem and progenitor cell development by mediating inflammatory signals in the niche.

The commensal microbiota regulates the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. Whether and how the microbiota influences HSPC development during embryogenesis is unclear. Using gnotobiotic zebrafish, we show that the microbiota is necessary for HSPC development and differentiation. Individual bacterial strains differentially affect HSPC formation, independent of their effects on myeloid cells. Early-life dysbiosis in chd8-/- zebrafish impairs HSPC development. Wild-type microbiota promote HSPC development by controlling basal inflammatory cytokine expression in kidney niche, and chd8-/- commensals elicit elevated inflammatory cytokines that reduce HSPCs and enhance myeloid differentiation. We identify an Aeromonas veronii strain with immuno-modulatory activities that fails to induce HSPC development in wild-type fish but selectively inhibits kidney cytokine expression and rebalances HSPC development in chd8-/- zebrafish. Our studies highlight the important roles of a balanced microbiome during early HSPC development that ensure proper establishment of lineal precursor for adult hematopoietic system.

Tox4 regulates transcriptional elongation and reinitiation during murine T cell development.

HMG protein Tox4 is a regulator of PP1 phosphatases with unknown function in development. Here we show that Tox4 conditional knockout in mice reduces thymic cellularity, partially blocks T cell development, and decreases ratio of CD8 to CD4 through decreasing proliferation and increasing apoptosis of CD8 cells. In addition, single-cell RNA-seq discovered that Tox4 loss also impairs proliferation of the fast-proliferating double positive (DP) blast population within DP cells in part due to downregulation of genes critical for proliferation, notably Cdk1. Moreover, genes with high and low expression level are more dependent on Tox4 than genes with medium expression level. Mechanistically, Tox4 may facilitate transcriptional reinitiation and restrict elongation in a dephosphorylation-dependent manner, a mechanism that is conserved between mouse and human. These results provide insights into the role of TOX4 in development and establish it as an evolutionarily conserved regulator of transcriptional elongation and reinitiation.