mTORC1 signaling pathway regulates tooth repair

Tooth germ injury can lead to abnormal tooth development and even tooth loss,affecting various aspects of the stomatognathic system including form,function,and appearance.However,the research about tooth germ injury model on cellular and molecule mechanism of tooth germ repair is still very limited.Therefore,it is of great importance for the prevention and treatment of tooth germ injury to study the important mechanism of tooth germ repair by a tooth germ injury model.Here,we constructed a Tg(dlx2b:Dendra2-NTR)transgenic line that labeled tooth germ specifically.Taking advantage of the NTR/Mtz system,the dlx2b+tooth germ cells were depleted by Mtz effectively.The process of tooth germ repair was evaluated by antibody staining,in situ hybridization,Ed U staining and alizarin red staining.The severely injured tooth germ was repaired in several days after Mtz treatment was stopped.In the early stage of tooth germ repair,the expression of phosphorylated 4E-BP1 was increased,indicating that mTORC1 is activated.Inhibition of mTORC1 signaling in vitro or knockdown of mTORC1 signaling in vivo could inhibit the repair of injured tooth germ.Normally,mouse incisors were repaired after damage,but inhibition/promotion of mTORC1 signaling inhibited/promoted this repair progress.Overall,we are the first to construct a stable and repeatable repair model of severe tooth germ injury,and our results reveal that mTORC1 signaling plays a crucial role during tooth germ repair,providing a potential target for clinical treatment of tooth germ injury.

Alterations in immune cell heterogeneities in the brain of aged zebrafish using single-cell resolution

Immunocytes,including the microglia,are crucial in the neurodegenerative process in old people.However,the understanding regarding microglia heterogeneity and other involved immunocytes remains elusive.We analyzed 26,456 immunocytes from 12-and 26-month-old zebrafish brains at single-cell resolution.Microglia and T lymphocytes were detected in the brain at both time points.Two types of microglia were annotated,namely,ac+microglia and xr+microglia,which were clustered into subsets 1,2,3,4,5,and subsets 6,7,8,9,respectively.Diversified microglia predominated the adult brains and cooperated with T cells to perform the functions of immune response and neuronal nutrition.We validated the specific microglia markers.The novel transgenic lines,Tg(lgals3bpb:e GFP)and Tg(apoc1:e GFP),were created,which faithfully labeled ac+microglia and served as valuable labeling tools.However,the microglia population reduced while T cells of six subtypes intriguingly increased to serve as the primary immune cells in aged brains.Unlike in 12-month-old brains,T cells,together with microglia,exhibited a coordinated signature of inflammation in the 26-month-old brains.Our findings revealed the immunocytes atlas in aged zebrafish brains.It implied the involvement of microglia and T cells in the progression of neurodegeneration in aging.

Zebrafish ppp1r21 mutant as a model for the study of primary biliary cholangitis

Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease that progresses to fibrosis and cirrhosis, resulting from the gradual destruction of intrahepatic bile ducts. Exploring genetic variants associated with PBC is essential to understand the pathogenesis of PBC. Here we identify a zebrafish balloon dog (blg) mutant with intrahepatic bile duct branching defects, exhibiting several key pathological PBC-like features, including immunodominant autoantigen PDC-E2 production, cholangiocyte apoptosis, immune cell infiltration, inflammatory activation, and liver fibrosis. blg encodes the protein phosphatase 1 regulatory subunit 21 (Ppp1r21), which is enriched in the liver and its peripheral tissues and plays a vital role in the early intrahepatic bile duct formation stage. Further studies show an excessive activation of the PI3K/AKT/mTOR pathway in the hepatic tissues in the mutant, while treatment with the pathway inhibitor LY294002 and rapamycin partially rescues intrahepatic bile duct branching defects and alleviates the PBC-like symptoms. These findings implicate the potential role of the Ppp1r21-mediated PI3K/AKT/mTOR pathway in the pathophysiology of PBC.

Zebrafish hhex-null mutant develops an intrahepatic intestinal tube due to de-repression of cdx1b and pdx1

The hepatopancreatic duct (HPD) system links the liver and pancreas to the intestinal tube and is composed of the extrahepatic biliary duct, gallbladder, and pancreatic duct. Haematopoietically expressed-homeobox (Hhex) protein plays an essential role in the establishment of HPD;however, the molecular mechanism remains elusive. Here, we show that zebrafish hhex-null mutants fail to develop the HPD system characterized by lacking the biliary marker Annexin A4 and the HPD marker sox9b. The hepatobiliary duct part of the mutant HPD system is replaced by an intrahepatic intestinal tube characterized by expressing the intestinal marker fatty acid-binding protein 2a (fabp2a). Cell lineage analysis showed that this intrahepatic intestinal tube is not originated from hepatocytes or cholangiocytes. Further analysis revealed that cdx1b and pdx1 are expressed ectopically in the intrahepatic intestinal tube and knockdown of cdx1b and pdx1 could restore the expression of sox9b in the mutant. Chromatin-immunoprecipitation analysis showed that Hhex binds to the promoters of pdx1 and cdx1b genes to repress their expression. We therefore propose that Hhex, Cdx1b, Pdx1, and Sox9b form a genetic network governing the patterning and morphogenesis of the HPD and digestive tract systems in zebrafish.

Ribosome biogenesis protein Urb1 acts downstream of mTOR complex 1 to modulate digestive organ development in zebrafish

Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in zebrafish urb1, which encodes an essential ribosome biogenesis protein. The urb1 cq31 mutant exhibits hypoplastic digestive organs, which is caused by impaired cell proliferation with the differentiation of digestive organ progenitors unaffected. Knockdown of mtor or raptor leads to similar hypoplastic phenotypes and reduced expression of urb1 in the digestive organs. Overexpression of Urb1 results in overgrowth of digestive organs, and can efficiently rescue the hypoplastic liver and pancreas in the mtor and raptor morphants. Reduced syntheses of free ribosomal subunits and impaired assembly of polysomes are observed in the urb1 mutant as well as in the mtor and raptor morphants, which can be rescued by the Urb1 overexpression. These data demonstrate that Urb1 plays an important role in governing ribosome biogenesis and protein synthesis downstream of mammalian/mechanistic target of rapamycin complex 1（mTORC1）, thus regulating the development of digestive organs. Our study indicates the requirement of hyperactive protein synthesis for the digestive organ development.

Hepatocytes in a normal adult liver are derived solely from the embryonic hepatocytes

In vertebrates,body weight increases many folds as a consequence of body growth from the childhood to adulthood（e.g.,～20 folds for a mouse）.Considering the fact that the liver-to-body weight ratio（LBR）stays relatively constant within species（Weglarz and Sandgren,2000;Kan et al.,2009）,the cell number in a liver must therefore keep increasing along with the growth of an individual organism. For example, during the growth of the zebrafish from 5 days post-fertilization （dpf） to 1.5 years old, the number of liver cells increased -900 folds （Figs. 1A and S1 ）.

The CXCR4-CXCL12 axis promotes T cell reconstitution via efficient hematopoietic immigration

T cells play a critical role in immunity to protect against pathogens and malignant cells.T cell immunodeficiency is detrimental,especially when T cell perturbation occurs during severe infection,irradiation,chemotherapy,and age-related thymic atrophy.Therefore,strategies that enhance T cell reconstitution provide considerable benefit and warrant intensive investigation.Here,we report the construction of a T cell ablation model in Tg(coro1a:Den NTR)zebrafish via metronidazole administration.The nascent T cells are mainly derived from the hematopoietic cells migrated from the kidney,the functional homolog of bone marrow and the complete recovery time is 6.5 days post-treatment.The cxcr4b gene is upregulated in the responsive hematopoietic cells.Functional interference of CXCR4 via both genetic and chemical manipulations does not greatly affect T lymphopoiesis,but delays T cell regeneration by disrupting hematopoietic migration.In contrast,cxcr4b accelerates the replenishment of hematopoietic cells in the thymus.Consistently,Cxcl12b,a ligand of Cxcr4,is increased in the thymic epithelial cells of the injured animals.Decreased or increased expression of Cxcl12b results in compromised or accelerated T cell recovery,respectively,similar to those observed with Cxcr4b.Taken together,our study reveals a role of CXCR4-CXCL12 signaling in promoting T cell recovery and provides a promising target for the treatment of immunodeficiency due to T cell injury.

Generation of Hematopoietic Stem Cells from Purified Embryonic Endothelial Cells by a Simple and Efficient Strategy

Recent progress by versatile approaches supports the new hypothesis that multi-potent hematopoietic stein cells （HSCs） are directly formed from a rare population of endothelial cells in mid-gestation mouse embryos. This process is therefore known as the endothelial-to- hematopoietic transition （EHT）. Nevertheless, there is no functional evidence that documents the HSC transition from purified endothelial cells. In this study, we developed an OP9-DLl-based co-culture system that was able to facilitate the HSC specification and/or expansion in vitro of mouse embryonic day 10.5 （El0.5） Tie2~ cells remarkably. Then, the immunophenotypically defined endothelial ceils were harvested by a combination of surface markers （Flkl＋CD31 ~CD41 CD45 Ter119 ） from the caudal half of EI0.0-EI 1.0 mouse embryos. The transplantation of the endothelia/OP9-DL1 co-cultures led to long-term, high-level, multi-lineage, and multi-organ he- matopoietic reconstitution in the irradiated adult recipients. The induced HSC activity was initially observed at El0.5, and a significant increase was detected at El 1.0, which suggests a temporally specific regulation. Taken together, tbr the first time, we provide functional evidence showing the HSC potential of purified embryonic endothelial cells, which is indispensable for the emerging EHT concept. Moreover, the newly defined co-culture system will aid the exploration of the key molecules governing the HSC transition from embryonic and even postnatal endothelial cells, which has enormous significance in basic and translational research.

Caudal dorsal artery generates hematopoietic stem and progenitor cells via the endothelial-to-hematopoietic transition in zebrafish

Zebrafish hematopoietic stem and progenitor cells（HSPCs） originate from the hemogenic endothelium of the ventral wall of the dorsal aorta（DA） through the endothelial-to-hematopoietic transition（EHT） from approximately 30 to 60 hours post fertilization（hpf）. However, whether other artery sites can generate HSPCs de novo remains unclear. In this study, using live imaging and lineage tracing, we found that the caudal dorsal artery（CDA） in the caudal hematopoietic tissue directly gave rise to HSPCs through EHT.This process initiated from approximately 60 hpf and terminated at approximately 156 hpf. Compared with that in the DA, fewer EHT events were observed in the CDA. The EHT events in the DA and CDA were similarly regulated by Runx1 but differentially influenced by blood flow（i.e., the EHT frequency in CDA was affected to a lesser extent when circulation was compromised in the tnnt2a～（-/-）mutant）. Therefore,the whole artery, including both DA and CDA, was endowed with the ability to produce HSPCs during a much longer time period. Coincidently, the lineage tracing results indicated that adult hematopoietic cells originated from the embryonic endothelium, and those produced later preferentially colonized the adult thymus. Collectively, our study revealed that the CDA serves as an additional source of hematopoiesis, and it shows similar but not identical properties with the DA.

GoldenFish:a rapid and efficient system to customize constructs for zebrafish transgenesis

Transgenesis, which inserts exogenous DNA into animal genomes, is a widely used technique. Traditionally, the constructs for transgenesis are generated by step-by-step subcloning of DNA fragments, which requires multiple steps depending on the construct complexity. To overcome the limitation, advanced tools such as Gateway cloning (Hartley et al., 2000;Kwan et al., 2007), In-Fusion cloning (Sleight et al., 2010), and Gibson assembly (Gibson et al., 2009) have been developed. However, due to their ligation characteristics, no systematic method for transgenesis has been developed. ‘Golden Gate’ cloning first appeared in 2008, which is a widely used DNA assembly method (Engler et al., 2008, 2009). Here, we take zebrafish transgenesis as an example and develop a standardized system called GoldenFish, which is based on Golden Gate cloning. It can customize transgenic constructs in one step and can be applied to multiple types of transgenesis such as one gene driven by one promoter, multiple genes driven by one promoter, and multiple genes respectively driven by multiple promoters, significantly reducing working time.