Stat3 Has a Different Role in Axon Growth During Development Than It Does in Axon Regeneration After Injury.

Signal transducer and activator of transcription 3 (STAT3) is essential for neural development and regeneration as a key transcription factor and mitochondrial activator. However, the mechanism of Stat3 in axon development and regeneration has not been fully understood. In this study, using zebrafish posterior lateral line (PLL) axons, we demonstrate that Stat3 plays distinct roles in PLL axon embryonic growth and regeneration. Our experiments indicate that stat3 is required for PLL axon extension. In stat3 mutant zebrafish, the PLL axon ends were stalled at the level of the cloaca, and expression of stat3 rescues the PLL axon growth in a cell-autonomous manner. Jak/Stat signaling inhibition did not affect PLL axon growth indicating Jak/Stat was dispensable for PLL axon growth. In addition, we found that Stat3 was co-localized with mitochondria in PLL axons and important for the mitochondrial membrane potential and ATPase activity. The PLL axon growth defect of stat3 mutants was mimicked and rescued by rotenone and DCHC treatment, respectively, which suggests that Stat3 regulates PLL axon growth through mitochondrial Stat3. By contrast, mutation of stat3 or Jak/Stat signaling inhibition retarded PLL axon regeneration. Meanwhile, we also found Schwann cell migration was also inhibited in stat3 mutants. Taken together, Stat3 is required for embryonic PLL axon growth by regulating the ATP synthesis efficiency of mitochondria, whereas Stat3 stimulates PLL axon regeneration by regulating Schwann cell migration via Jak/Stat signaling. Our findings show a new mechanism of Stat3 in axon growth and regeneration.

CircRNA-CIRH1A Promotes the Development of Osteosarcoma by Regulating PI3K/AKT and JAK2/STAT3 Signaling Pathways.

Osteogenic sarcoma (OS), one of the mesenchymal tumors with a high degree of malignancy, mainly occurs in the metaphysis of the long bones and around the knee joints in children and adolescents. The poor diagnosis in patients with OS can be attributed to the lack of early clinical symptoms, although the growth of tumor mass gradually results in severe pain and systemic symptoms. The mechanisms underlying the pathogenesis of OS are not fully understood. Thus, identifying early diagnostic biomarkers and novel targets involved in the progression of OS is of critical significance in the management of OS. CircRNA is a class of non-coding RNAs characterized by the close-loop structure and increased stability, which are implicated in the regulation of cell proliferation, differentiation, migration, and apoptosis. Moreover, circRNAs also play significant roles in aging and chronic disorders, such as cancer and cardiovascular diseases. Accordingly, we reported the upregulation of circRNA-CIRH1A in OS tissues and cell lines. Silencing circRNA-CIRH1A in OS cell lines (U2OS, HOS, Saos-2, and MG-63) could inhibit the cell proliferation, invasion, migration, and apoptosis, which was also validated in xenograft tumorigenesis mouse model. We further demonstrated that circRNA-CIRH1A sponged miR-1276, which subsequently disrupted the effect of miR-1276 on PI3K/AKT and JAK2/STAT3 signaling pathways. Together, our study revealed the oncogenic role of circRNA-CIRH1A in OS, and identified miR-1276/ PI3K-AKT and JAK2-STAT3 signaling axis as the key downstream mediators of circRNA-CIRH1A.

Hemifacial microsomia is linked to a rare homozygous variant V162I in FRK and validated in zebrafish.

OBJECTIVES: Hemifacial microsomia (HFM) is a common birth defect involving the first and second branchial arch derivatives. Although several chromosomal abnormalities and causal gene variants have been identified, genetic etiologies in a majority of cases with HFM remain unknown. This study aimed to identify genetic mutations in affected individuals with HFM. METHODS: Whole-exome sequencing and bioinformatics analysis were performed for 16 affected individuals and their family members. Sanger sequencing was applied for confirmation of selected mutations. Zebrafish embryos were used for in situ hybridization of candidate gene, microinjection with antisense morpholino, and cartilage staining. RESULTS: A homozygous missense mutation (c.484G > A; p.V162I) in the FRK gene was identified in an 18-year-old girl with HFM and dental abnormalities. Heterozygous mutation of this mutation was identified in her parents, who are first cousins in a consanguineous family. FRK is highly expressed in the Meckel's cartilage during embryonic development in mouse and zebrafish. Knockdown of frk in zebrafish showed a lower length and width ratio of Meckel's cartilage, abnormal mandibular jaw joint, and disorganized ceratobranchial cartilage and bone. CONCLUSIONS: We identified a recessive variant in the FRK gene as a novel candidate gene for a patient with HFM and mandibular hypoplasia and revealed its effects on craniofacial and embryonic development in zebrafish.

"Omics" data unveil early molecular response underlying limb regeneration in the Chinese mitten crab, Eriocheir sinensis.

Limb regeneration is a fascinating and medically interesting trait that has been well preserved in arthropod lineages, particularly in crustaceans. However, the molecular mechanisms underlying arthropod limb regeneration remain largely elusive. The Chinese mitten crab Eriocheir sinensis shows strong regenerative capacity, a trait that has likely allowed it to become a worldwide invasive species. Here, we report a chromosome-level genome of E. sinensis as well as large-scale transcriptome data during the limb regeneration process. Our results reveal that arthropod-specific genes involved in signal transduction, immune response, histone methylation, and cuticle development all play fundamental roles during the regeneration process. Particularly, Innexin2-mediated signal transduction likely facilitates the early stage of the regeneration process, while an effective crustacean-specific prophenoloxidase system (ProPo-AS) plays crucial roles in the initial immune response. Collectively, our findings uncover novel genetic pathways pertaining to arthropod limb regeneration and provide valuable resources for studies on regeneration from a comparative perspective.

Dataset for transcriptomic, H3K9ac and H3K9me3 profiles during cardiac regeneration.

Acetylation and tri-methylation of histone H3 lysine 9 (H3K9ac and H3K9me3) play an interactive regulatory role in the epigenetic regulation of gene expression during heart development and cardiovascular disease, but little is known about their possible role in heart regeneration. Here we utilized genome-wide high-throughput RNA sequencing (RNA-seq) and chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) for H3K9ac and H3K9me3, carried out on regenerative cardiac tissues at different days post amputation in zebrafish (Danio rerio) to investigate dynamic changes in gene expression and the epigenetic landscape of H3K9ac and H3K9me3. The STAR, Bowtie2, MACS2, and deepTools2 were mainly used for RNA-Seq or ChIP-seq data analysis. In this article, we present detailed information on experiment design, data generation, quality assessment and processing pipeline. Raw reads of the RNA-seq and ChIP-seq data have been deposited at the NCBI GEO repository with the accession number GSE158104. Our data will be a valuable resource for the elucidation of H3K9ac and H3K9me3 involvement in the regulation of gene transcription during cardiac regeneration.

Reorganization of 3D chromatin architecture in doxorubicin-resistant breast cancer cells.

Background: Doxorubicin resistance remains a major therapeutic challenge leading to poor survival prognosis and treatment failure in breast cancer. Although doxorubicin induces massive changes in the transcriptional landscape are well known, potential diagnostic or therapeutic targets associated with the reorganization of three-dimensional (3D) chromatin architecture have not yet been systematically investigated. Methods: Here we performed in situ high-throughput chromosome conformation capture (Hi-C) on parental and doxorubicin-resistant MCF7 (MCF7-DR) human breast cancer cells, followed by integrative analysis of HiC, ATAC-seq, RNA-seq and TCGA data. Results: It revealed that A/B compartment switching was positively correlated to genome-wide differential gene expression. The genome of MCF7-DR cells was spatially reorganized into smaller topologically associating domains (TADs) and chromatin loops. We also revealed the contribution of increased chromatin accessibility and potential transcription factor families, including CTCF, AP-1 and bHLH, to gained TADs or loops. Intriguingly, we observed two condensed genomic regions (∼20 kb) with decreased chromatin accessibility flanking TAD boundaries, which might play a critical role in the formation or maintenance of TADs. Finally, combining data from TCGA, we identified a number of gained and lost enhancer-promoter interactions and their corresponding differentially expressed genes involved in chromatin organization and breast cancer signaling pathways, including FA2H, FOXA1 and JRKL, which might serve as potential treatment targets for breast cancer. Conclusion: These data uncovered a close connection between 3D genome reorganization, chromatin accessibility as well as gene transcription and provide novel insights into the epigenomic mechanisms involving doxorubicin resistance in breast cancer.

Keratin5-cytoskeleton-BMP4 network regulates cell phenotype conversions during cardiac regeneration.

The reconstruction of a blood supply system and myocardial recovery from inflamamtory reactions in the infract zone remains a challenge in cardiac regeneration after myocardial infarction. Here, we observed that the local myocardial cells and the clotted blood cells undergo cellular remodeling via cytoplasmic exocytosis and nuclear reorganization in zebrafish hearts after resection of the ventricular apex. The subsequent tissue regeneration processes were visualized by detection of the spatiotemporal expression of three tissue specific genes (α-SMA which marks for vasculature/fibrogenesis, Flk1for angiogenesis/hematopoiesis, and Pax3a for remusculogensis), and two histone modification markers (H3K9Ac and H3K9Me3 for chromatin remodeling). By analyzing the composition of the blastema tissue fractions we found that Krt5 peptide could promote F-actin assembly, BMP4-pSmad2/5/8 signaling activity, and H3K9Me3-mediated chromatin accessibility at the blastema representative genes in the cultured zebrafish embryonic fibroblasts. Further in vivo tests demonstrated that Krt5 interacted with beta actin, and promoted Gata3 expression and Flk1-GFP marked blastema angiogenesis. These results proposed a new Krt5-cytoskeleton-BMP4 mechanotransduction mechanism in the epithelial-dependent and cell phenotype conversion-based tissue regeneration.

Mesenchymal Stem Cell-Derived Extracellular Vesicle-Shuttled microRNA-302d-3p Represses Inflammation and Cardiac Remodeling Following Acute Myocardial Infarction.

Our research intended to investigate the roles of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) in acute myocardial infarction (AMI) via delivery of microRNA (miR)-302d-3p. AMI mouse models were established. EVs isolated from MSCs with miR-302d-3p mimic were injected near the infarct area or co-cultured with hypoxic cardiomyocytes to evaluate their effects. The expression of NF-κB pathway-related genes and inflammatory factors was determined. AMI mice exhibited downregulated miR-302d-3p and elevated MD2 and BCL6 levels. BCL6 was negatively targeted by miR-302d-3p and could bind to MD2 promoter to upregulate MD2 expression. MSCs-EVs, MSCs-EVs carrying miR-302d-3p, or BCL6 or MD2 silencing inactivated the NF-κB pathway and alleviated infarcted area, myocardial fibrosis, inflammation, apoptosis, and cardiac dysfunction in AMI mice. Besides, MSCs-EVs, MSCs-EVs carrying miR-302d-3p, or BCL6 or MD2 silencing diminished viability and inflammation but augmented apoptosis of hypoxic cardiomyocytes. Conclusively, MSCs-EVs carrying miR-302d-3p repressed inflammation and cardiac remodeling after AMI via BCL6/MD2/NF-κB axis.

Prophage-encoded gene VpaChn25_0734 amplifies ecological persistence of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is a waterborne pathogen that can cause acute gastroenteritis, wound infection, and septicemia in humans. The molecular basis of its pathogenicity is not yet fully understood. Phages are found most abundantly in aquatic environments and play a critical role in horizontal gene transfer. Nevertheless, current literature on biological roles of prophage-encoded genes remaining in V. parahaemolyticus is rare. In this study, we characterized one such gene VpaChn25_0734 (543-bp) in V. parahaemolyticus CHN25 genome. A deletion mutant ΔVpaChn25_0734 (543-bp) was obtained by homologous recombination, and a revertant ΔVpaChn25_0734-com (543-bp) was also constructed. The ΔVpaChn25_0734 (543-bp) mutant was defective in growth and swimming mobility particularly at lower temperatures and/or pH 7.0-8.5. Cell surface hydrophobicity and biofilm formation were significantly decreased in the ΔVpaChn25_0734 (543-bp) mutant (p < 0.05). Based on the in vitro Caco-2 cell model, the deletion of VpaChn25_0734 (543-bp) gene significantly reduced the cytotoxicity of V. parahaemolyticus CHN25 to human intestinal epithelial cells (p < 0.05). Comparative secretomic and transcriptomic analyses revealed a slightly increased extracellular proteins, and thirteen significantly changed metabolic pathways in the ΔVpaChn25_0734 (543-bp) mutant, showing down-regulated carbon source transport and utilization, biofilm formation, and type II secretion system (p < 0.05), consistent with the observed defective phenotypes. Taken, the prophage-encoded gene VpaChn25_0734 (543-bp) enhanced V. parahaemolyticus CHN25 fitness for survival in the environment and the host. The results in this study facilitate better understanding of pathogenesis and genome evolution of V. parahaemolyticus, the leading sea foodborne pathogen worldwide.

Integrated Chromatin Accessibility and Transcriptome Landscapes of Doxorubicin-Resistant Breast Cancer Cells.

BACKGROUND: Doxorubicin is one of the most effective chemotherapeutic drugs for breast cancer while its common drug resistance leads to poor patient prognosis and survival. Growing evidence indicate dynamically reorganized chromatin allows rapid access of the gene regulatory machinery to open genomic regions facilitating subsequent gene expression through direct transcription factor (TF) activation and regulatory element binding. METHODS: To better understand the regulatory network underlying doxorubicin resistance in breast cancer cells, we explored the systematic alterations of chromatin accessibility and gene expression by the assay for transposase-accessible chromatin using sequencing (ATAC-seq) in combination with RNA sequencing, followed by integrative analysis to identify potential regulators and their targets associated with differentially accessible regions (DARs) in doxorubicin-resistant MCF7 (MCF7-DR) cells. RESULTS: A total of 3,963 differentially expressed genes (DEGs) related to doxorubicin resistance were identified, including dramatically up-regulated MT1E, GSTP1, LDHB, significantly down-regulated TFF1, UBB, DSCAM-AS1, and histone-modifying enzyme coding genes HDAC2, EZH2, PRMT5, etc. By integrating with transcriptomic datasets, we identified 18,228 DARs in MCF7-DR cells compared to control, which were positively correlated with their nearest DEGs (r = 0.6). There were 11,686 increased chromatin-accessible regions, which were enriched in up-regulated genes related to diverse KEGG pathways, such as the cell cycle, regulation of actin cytoskeleton, signaling pathways of MAPK, PI3K/Akt and Hippo, which play essential roles in regulating cell apoptosis, proliferation, metabolism, and inflammatory responses. The 6,542 decreased chromatin-accessible regions were identified for the declined doxorubicin-associated biological processes, for instance, endocrine and insulin resistance, central carbon metabolism, signaling pathways of TGF-beta and P53. Combining data from TCGA, analyses of the DAR sequences associated with the DNA-binding motifs of significantly enriched TF families including AP-1, TEAD and FOX, indicated that the loss-function of FOXA1 might play a critical role in doxorubicin-resistant breast cancer cells (DOX-R BCCs). CONCLUSION: These data exhibit the non-genetic landscape of chromatin accessibility and transcript levels in the DOX-R BCCs, and provide clear insights and resources for the detection of critical TFs and potential cis-regulatory elements-based putative therapeutic targets.

Prophage-Related Gene VpaChn25_0724 Contributes to Cell Membrane Integrity and Growth of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is a leading seafood-borne pathogen that can cause acute gastroenteritis and even death in humans. In aquatic ecosystems, phages constantly transform bacterial communities by horizontal gene transfer. Nevertheless, biological functions of prophage-related genes in V. parahaemolyticus remain to be fully unveiled. Herein, for the first time, we studied one such gene VpaChn25_0724 encoding an unknown hypothetical protein in V. parahaemolyticus CHN25. This gene deletion mutant ΔVpaChn25_0724 was constructed by homologous recombination, and its complementary mutant ΔVpaChn25_0724-com was also obtained. The ΔVpaChn25_0724 mutant exhibited a sever defect in growth and swimming motility particularly at lower temperatures. Biofilm formation and cytotoxicity capacity of V. parahaemolyticus CHN25 was significantly lowered in the absence of VpaChn25_0724. Comparative secretomic analysis revealed an increase in extracellular proteins of ΔVpaChn25_0724, which likely resulted from its damaged cell membrane. Comparison of transcriptome data showed twelve significantly altered metabolic pathways in ΔVpaChn25_0724, suggesting inactive transport and utilization of carbon sources, repressed energy production and membrane biogenesis in ΔVpaChn25_0724. Comparative transcriptomic analysis also revealed several remarkably down-regulated key regulators in bacterial gene regulatory networks linked to the observed phenotypic variations. Overall, the results here facilitate better understanding of biological significance of prophage-related genes remaining in V. parahaemolyticus.

Specific miRNA-G Protein-Coupled Receptor Networks Regulate Sox9a/Sox9b Activities to Promote Gonadal Rejuvenation in Zebrafish.

Fertility and endocrine function rely on a tightly regulated synchronicity within the hypothalamic-pituitary-gonadal axis, for which the sex gonad serves as the primary source of sex steroid hormones and germ cells. To maintain hormonal stasis and fertility throughout the lifespan, inducing gonadal stem cell renewal is an attractive strategy. The follicle-stimulating hormone/cAMP/MAPK/Sox9 signaling axis and its regulated specific miRNAs are thought to regulate vertebrate gonadal development and sex differentiation, yet the regulatory networks are largely unknown. By genome-wide transcriptome mining and gonadal microinjections, we identify two G protein-coupled receptor (GPCR)-regulatory circuits: miR430a-Sox9a in the testis and miR218a-Sox9b in the ovary. Coinjection of a Sox9a-miR430a mixture promotes spermatogenesis, whereas Sox9b-miR218a mixture increases primordial ovarian follicles. Coimmunoprecipitation and mass spectrometry indicate that the two mixtures differentially modulate Sox9a/Sox9b multiple covalent modifications. We further reveal that miR430a and Sox9a synergistically activate testicular protein kinase C (PKC)/Akt signaling, whereas the miR218a and Sox9b mixture constrains ovary PKC/Akt signaling. pMIR-GFP reporter assay demonstrate that miR430a and miR218a target the 3' untranslated region (UTR) of four GPCR targets (lgr4, grk5l, grk4, and grp157). Knockdown of these GPCR genes or two Sox9 genes alters miR430a and miR218a regulation in the above gonad-specific PKC and Akt signaling pathways. These results establish two specific miRNA-GPCR-Sox9 networks and provide mechanistic insight into gonadal differentiation and rejuvenation. Stem Cells 2019;37:1189-1199.

Dietary Safety Assessment of Flk1-Transgenic Fish.

Genetic engineering, also called genetic modification, is facing with growing demands of aquaculture and aquatic products. Although various genetically modified (GM) aquatics have been generated, it is important to evaluate biosafety of GM organisms on the human health before entering into our food chain. For this purpose, we establish a zebrafish wild adult feeding Flk1-transgenic larvae model to examine the predatory fish's histology in multiple tissues, and the global gene expression profile in the liver. 180 days of feeding trial show that there are no significantly morphological changes in intestine, liver, kidney, and sex gonads between fish fed with Flk1 transgenic fish diet (TFD) and fish fed with regular food meal (RFM). However, a characteristic skin spot and autofluorescence increase in the theca of follicle are observed in F1 generation of TFD fish. Liver RNA-sequencing analyses demonstrate that 53 out of 56712 genes or isoforms are differentially transcribed, and mostly involved in proteolysis in extracellular region. According to GO enrichment terms these deregulated genes function in catalytic activity, steroid storing, lipid metabolic process and N-Glycan biosynthesis. These results suggest that a long term of Flk1-transgenic fish diet could alter certain metabolic pathways and possibly cause related tissue deformation. Compared to the previous reports, our feasible transgenic dietary assess system could evaluate subchronic and potential health impact of transgenic fish diet by combining multi-tissue histology and liver transcriptome analyses.

Tissue Extract Fractions from Starfish Undergoing Regeneration Promote Wound Healing and Lower Jaw Blastema Regeneration of Zebrafish.

Natural bioactive materials provide an excellent pool of molecules for regenerative therapy. In the present study, we amputate portions of the arms of Archaster typicus starfish, extract and separate the active biomaterials, and compare the effects of each fraction on in vitro wound healing and in vivo lower jaw regeneration of zebrafish. Compared with crude extract, normal hexane fractions (NHFs) have a remarkable effect on cellular proliferation and collective migration, and exhibit fibroblast-like morphology, while methanol-water fractions (MWFs) increase cell size, cell-cell adhesion, and cell death. Relative to moderate mitochondrialand lysosomal aggregation in NHFs-cultured cells, MWFs-cultured cells contain more and bigger lysosomal accumulations and clump detachment. The in vivo zebrafish lower jaw regeneration model reveals that NHFs enhance blastema formation and vasculogenesis, while MWFs inhibit fibrogenesis and induce cellular transformation. Gene expression analyses indicate that NHFs and MWFs separately activate blastema-characteristic genes as well as those genes-related to autophagy, proteasome, and apoptosis either during cell scratch healing or ganciclovir-induced apoptosis. Our results suggest that bioactive compounds from NHFs and MWFs could induce blastema formation and remodeling, respectively, and prevent tissue overgrowth.

Menin localization in cell membrane compartment.

Menin is encoded by the MEN1 gene, which is mutated in an inherited human syndrome, multiple endocrine neoplasia type 1(MEN1). Menin is primarily nuclear protein, acting as a tumor suppressor in endocrine organs, but as an oncogenic factor in the mixed lineage leukemia, in a tissue-specific manner. Recently, the crystal structures of menin with different binding partners reveal menin as a key scaffold protein that functionally interacts with various partners to regulate gene transcription in the nucleus. However, outside the nucleus, menin also regulates multiple signaling pathways that traverse the cell surface membrane. The precise nature regarding to how menin associates with the membrane fraction is poorly understood. Here we show that a small fraction of menin associates with the cell membrane fraction likely via serine palmitoylation. Moreover, the majority of the membrane-associated menin may reside inside membrane vesicles, as menin is protected from trypsin-mediated proteolysis, but disruption of the membrane fraction using detergent abolishes the detection. Consistently, cellular staining for menin also reveals the distribution of menin in the cell membrane and the punctate-like cell organelles. Our findings suggest that part of intracellular menin associates with the cell membrane peripherally as well as resides within the membrane vesicles.

[Morphological and Molecular Identification of Acanthamoeba sp., A New Pathogen for Human Respiratory Tract Infection].

Objective: To identify the species of a morphologically Acanthamoeba-like pathogen in sputum from a patient with repeated cough. Methods: Protozoa were isolated from the sputum and cultured for morphological observation of the trophozoites and cysts. DNA was extracted from the cultivated sample, and PCR was performed using primers as follows: 18S universal primers for amoeba family（Ami6F1 and Ami9R） and for amoeba genus（JDP1 and JDP2）, and primers for 18S full-length sequence of S-7 ATCC reference strain of Acanthamoeba griffini （AacGF and AscGR）. The 18S rRNA was sequenced, followed by homology analysis. The maximum likelihood method was used to construct phylogenetic tree. Results: Microscopic examination showed that the trophozites had spine and irregular-shape pseudopodia bulge. The cysts were encapsulated by double membrane layer with the inner membrane having star-like processes. As expected, PCR amplification resulted in bands of 830, 479 and 1 957 bp, respectively, which were blasted to be 99%, 99% and 100% homologous to those of A. griffini（U07412.1）. Phylogenetic tree indicated that this acanthamobe in the patient's sample was 91.4%, 99.6%, 94.5% and 91.8% homologous to keratitis-associated A. castellanii, A. polyphage, A. cullbertsoni and A. rhysodes. Conclusion: The parasite in sputum of the patient with respiratory tract infection is Acanthamoeba griffini.

Tetraspanin CD82: a suppressor of solid tumors and a modulator of membrane heterogeneity.

Tetraspanin CD82 suppresses the progression and metastasis of a wide range of solid malignant tumors. However, its roles in tumorigenesis and hematopoietic malignancy remain unclear. Ubiquitously expressed CD82 restrains cell migration and cell invasion by modulating both cell-matrix and cell-cell adhesiveness and confining outside-in pro-motility signaling. This restraint at least contributes to, if not determines, the metastasis-suppressive activity and, also likely, the physiological functions of CD82. As a modulator of cell membrane heterogeneity, CD82 alters microdomains, trafficking, and topography of the membrane by changing the membrane molecular landscape. The functional activities of membrane molecules and the cytoskeletal interaction of the cell membrane are subsequently altered, followed by changes in cellular functions. Given its pathological and physiological importance, CD82 is a promising candidate for clinically predicting and blocking tumor progression and metastasis and also an emerging model protein for mechanistically understanding cell membrane organization and heterogeneity.

Time point-based integrative analyses of deep-transcriptome identify four signal pathways in blastemal regeneration of zebrafish lower jaw.

There has been growing interest in applying tissue engineering to stem cell-based regeneration therapies. We have previously reported that zebrafish can faithfully regenerate complicated tissue structures through blastemal cell type conversions and tissue reorganization. To unveil the regenerative factors and engineering arts of blastemal regeneration, we conducted transcriptomal analyses at four time points corresponding to preamputation, re-epitheliation, blastemal formation, and respecification. By combining the hierarchical gene ontology term network, the DAVID annotation system, and Euclidean distance clustering, we identified four signaling pathways: foxi1-foxo1b-pou3f1, pax3a-mant3a-col11/col2, pou5f1-cdx4-kdrl, and isl1-wnt11 PCP-sox9a. Results from immunohistochemical staining and promoter-driven transgenic fish suggest that these pathways, respectively, define wound epidermis reconstitution, cell type conversions, blastemal angiogenesis/vasculogenesis, and cartilage matrix-orientation. Foxi1 morpholino-knockdown caused expansions of Foxo1b- and Pax3a-expression in the basal layer-blastemal junction region. Moreover, foxi1 morphants displayed increased sox9a and hoxa2b transcripts in the embryonic pharyngeal arches. Thus, a Foxi1 signal switch is required to establish correct tissue patterns, including re-epitheliation and blastema formation. This study provides novel insight into a blastema regeneration strategy devised by epithelial cell transdifferentiation, blood vessel engineering, and cartilage matrix deposition.

Two origins of blastemal progenitors define blastemal regeneration of zebrafish lower jaw.

Zebrafish possess a remarkable ability to regenerate complicated structures by formation of a mass of undifferentiated mesenchymal cells called blastema. To understand how the blastema retains the original structural form, we investigate cellular transitions and transcriptional characteristics of cell identity genes during all stages of regeneration of an amputated lower jaw. We find that mesenchymal blastema originates from multiple sources including nucleated blood cells, fibroblasts, damaged muscle cells and pigment cells. These cells are transformed into two populations of blastemal progenitors: foxi1-expression and isl1-expression, before giving rise to cartilage, bone, and muscle. Time point- based transcriptomal analysis of 45 annotated Hox genes reveal that five 3'-end Hox genes and an equal number of 5'-end Hox genes are activated largely at the stage of blastema reformation. RNA in situ hybridization shows that foxi1 and pax3a are respectively expressed in the presumptive mandible skeletal region and regenerating muscle at 5 dpa. In contrast, hoxa2b and hoxa11b are widely expressed with different domain in chondrogenic blastema and blastema mesenchyme. Knockdown foxi1 changes the expression patterns of sox9a and hoxa2b in chondrogenic blastema. From these results we propose that two origins of blastemal progenitors define blastema skeleton and muscle respecifications through distinct signaling pathways. Meanwhile, the positional identity of blastema reformation is implicated in mesenchymal segmentation and characteristic expression pattern of Hox genes.

Using a yeast inverse one-hybrid system to identify functional binding sites of transcription factors.

Binding of transcription factors to promoters is a necessary step to initiate transcription. From an evolutionary standpoint, the regulatory proteins and their binding sites are considered to have molecularly coevolved. We developed an efficient yeast strategy, an "inverse one-hybrid system", to identify binding targets of transcription factors globally in a genome of interest. The technique consists of a yeast strain expressing a -transcription factor of interest mated to yeast containing a library of random genomic fragments cloned upstream of a reporter gene (URA3). Positive growth on media without uracil denotes a fragment being bound by the transcription factor, e.g., zebrafish FoxI1. The bound fragments in hundreds of positive clones are sequenced and retested for their binding activities using a colony PCR and sequencing strategy. The resulting tools allow for rapid and genomic-wide identification of transcriptional binding targets.