Exploring The Role Of Hhex In Liver Regeneration With A Newly-established High-resolution In Situ Expression Detection Technique For Multi-factors In Zebrafish Larvae

Part1:Whole-mount in situ hybridization（WISH）is widely applied to explore gene expression in many different organisms during embryogenesis,including Drosophila,Xenopus,zebrafish and mammals.The principle of WISH is that the specifically labeled nucleic acid probe and the target nucleic acid sequence could specifically anneal to complement each other in fixed tissues,and it can detect spatiotemporal expression of gene.WISH has been widely used in the study of the localization of gene expression in histocyte and different developmental stages.The in situ hybridization（ISH）first appeared in the 1960s.And since 1980s,this technique has been continuously developed and improved.The original technique relied on radiolabeled nucleic acid probes which had disadvantages that the sensitivity and spatial resolution cannot be obtained simultaneously.Meanwhile,radioactivity also has potential health risks.Non-radioactive RNA fluorescently labeled probes appeared in 1980s.Along with the technology progress,more technique came out which was faster,safer and more sensitive.The latest technique is the development of fluorescence in situ hybridization（FISH）by using tyramide signal amplification（TSA）technique,which makes the signal is more specific by taking advantage of a reactive fluorescent substrate that can quickly and covalently bind to peroxidase（POD）.Compared with WISH,the diffusion substrate of AP is generated in the covalent binding reaction,which makes FISH more sensitive so that the experimental result can achieve single-cell resolution under a confocal laser microscope.Whole-mount antibody staining/immunofluorescence（IF）detects antigens localization and expression by using fluorescently labeled secondary antibody coupled to the primary antibody to amplify the corresponding signal.Compared with ISH,the optical resolutions of IF is higher so that it can be used to accurately locate proteins even at the intracellular level and detect secreted proteins.In addition,the m RNA effective half-life is short but the protein is relatively stable,in which case detecting the protein expression level is more beneficial for studying gene expression patterns.However,the defect of IF is that it requires specific antibodies,which are more difficult to obtain than specific RNA probes for ISH.The combination of FISH and IF（FISH-IF）has been successfully applied to the detection of gene expression in Drosophila.But among the currently available methods,IF is carried out before FISH.Traditional ISH uses proteinase K to treat embryos to enhance the penetration of the probe which is at the expense of antigen integrity and not feasible at larvae stage.RNAscope technique has been developed in recent years,which is based on the target probe hybridization fluorescence signal amplification in situ RNA detection.RNAscope technique could help us to detect gene expression in early embryos and tissue section.Though the combination of FISH or RNAscope and IF has been applied for zebrafish,these techniques are limited to early embryos and tissue section,and is not applicable to the staining of larvae.As a crucial model organism to study human genetic disorders,zebrafish has outstanding advantages,such as transparent embryos at early stages,in vitro fertilization,short sexual maturity,large number of embryos produced,facilitate large-scale forward genetic mutagenesis to screen small molecule,and strong regeneration ability.Meanwhile,the zebrafish genome is 70%similar to human genome which is evolutionary conservative.Therefore,it has important biological sence to use zebrafish as a model organism to study important genes involved in organ development and damaged regeneration.ISH and IF technique is usually used to detect the zebrafish gene expression.But the types of reliable antibodies in zebrafish are very limited.From the later embryos stage（3-5dpf）and larvae（5dpf/120hpf）,RNA probes are so hard to penetrate the dense skin and deep tissue that detecting gene expression in whole-mount embryos become difficult.Because of the conditions,it is impossible to detect the multiple m RNAs concurrently with high-resolution which limits the superiority of zebrafish as model organism to study on late organogenesis and regeneration capabilities.To overcome the difficulty of detection gene expression in the later stage of zebrafish development,we optimized the FISH-IF so that it can be used to detect gene expression pattern in various tissues of different embryonic and larvae.Within this technique,FISH is operated before IF to prevent the m RNA in the tissue from being degraded during the IF experiment.Artificial skin removal and Triton X-100 treatment are used to replace traditional proteinase K to enhance embryo permeability while preserving intact epitopes.We use the probe template synthesized by PCR to synthesize RNA probes and avoid non-specific binding of probes in transgenic zebrafish.In addition,we use high-strength eluent to elute non-specifically bound RNA probes and reduce background signals.Both FISH and IF of this method will show fluorescent signals.Confocal is usually used as a detection method.Compared with traditional WISH,FISH-IF can simultaneously detect multiple m RNAs and proteins in whole zebrafish embryos with high sensitivity and high resolution（even single cell level）.Using the optimized technology,we detected gene expression in various organs at different stages in zebrafish,such as the expression patterns of the endoderm organs makers at 36hpf.The gene expression patterns of the digestive organs（such as liver,pancreas,intestine and bile duct）,cerebrovascular and lymphatic at 5dpf are clear.Meanwhile,we detected deep tissues（muscles,spinal cord and notochord）and superficial tissues（pectoral fin,skin and hair cells）from 4dpf to 10dpf.The expression of markers in the liver and bile duct at 20 dpf can be detected at latest.Our technique has higher resolution besides studying multiple genes expression so that we can locate the target m RNA at single-cell level.This method overcomes the shortcomings of traditional FISH-IF（such as low penetration rate in larvae and weak signal after proteinase K treatment）,thereby improving the sensitivity of detection.When the target m RNA is detected by FISH and using IF to co-stain the antibody can indicate the subcellular localization of the protein and provide more information about the gene expression pattern.The internal fluorescence of the fluorescent protein carried by the transgenic line is quenched during FISH when we detect gene expression.We can use the specific antibody that specifically recognizes the fluorescent protein to show the gene localization.Therefore,when there is no suitable antibody,we can use a transgenic strain that expresses fluorescent protein in the target tissue to detect the expression of the gene in the tissue.Using FISH-IF optimized technique which can be applied to zebrafish embryonic stage or whole embryos of larvae,we can detect three or four kinds of m RNA and protein simultaneously.This technique has high sensitivity and high resolution which can become a powerful technique for studying in situ gene expression in zebrafish.Part 2:The liver is a multifunctional digestive organ,which has the functions of protein synthesis,glycogen storage,amino acid secretion,blood storage and filtration.It is mainly composed of liver parenchymal cells and bile duct epithelial cells（BECs）,which are differentiated from liver progenitor cells during its development.In mammals,hepatocytes provide most of the functions related to body homeostasis,accounting for more than 80%of the liver mass.For some related diseases of the liver,such as liver cancer,the current effective treatment methods and methods are mainly through the removal of the cancerous liver,but this treatment method will cause the patient’s liver function to decline.The liver is now known to be one of the organs that can regenerate which can revert back to its original size and function after injury.The unique regenerative capacity of the liver guarantees the essential function of liver for most organisms.Therefore,it is very important to study the mechanism of liver regeneration.There are three main sources of liver cells in the process of liver regeneration.One is the self-proliferation of hypotocytes:the removal of two-thirds of the liver in rodents will be back to the origin weight through the proliferation of the remaining liver.The second is the differentiation of hepatic progenitor cells:when the liver is chronically damaged by drugs,the endogenous precursor cells will be activated and then differentiate into liver cells.The third is the transdifferentiation of bile duct cells:when the liver is severely damaged,the remaining hepatocytes will lose the ability of karyokinesis.At this moment mature bile duct cells can transdifferentiate into functional hepatocytes which will participate in liver regeneration.So far the important regulatory factors involved in the progress of bile duct cells transdeffrentiating into hepatocytes and their regulatory mechanisms are still unclear.Hematopoietically expressed homeobox（HHEX）,also known as proline-enriched homeodomain（PRH）,is a transcription factor and one of the earliest expression precursors of endothelium and hematopoiesis in Xenopus,zebrafish,and mice.The importance of HHEX in tumorigenesis has begun to be accepted widely.The change of HHEX levels and HHEX subcellular localization contribute to several cancers and certain subtypes of leukemia.HHEX has the ability to inhibit normal epithelial cell proliferation,migration and invasion of surrounding tissues,so the absence of HHEX is an important sign of many diseases.Many studies have shown that HHEX plays an important role in cell proliferation and differentiation.This gene can participate in the regulation during organogenesis such as blood cells,liver,and pancreas.In the mouse model,the HHEX mutant has multiple developmental defect phenotypes,including abnormal development of the heart,liver,and thyroid,and defect of angiogenesis.The phenotype of the liver is that the liver become smaller and cystic,without gallbladder and extrahepatic duct.This phenotype indicates that HHEX is necessary for the development of the liver and bile ducts.HHEX itself is regulated by HGF3β,SMAD,WNT,etc.,and can mediate the differentiation of stem cells into hepatocytes.Though many reports have shown that HHEX plays an important role in liver development and regeneration,there is no direct experimental evidence to show whether or not knock out of HHEX will affect liver regeneration.We use the transgenic line Tg（lfabp:Dendra2-NTR）which is based on MTZ/NTR system to specifically damage the liver,where we apply CRISPR/Cas9 technique to construct hhex knock out mutant.Then we can study the mechanism of hepatocytes regeneration in hhex mutant after MTZ treatment.However,hhex plays an important role in the early development of the liver and biliary system:homozygotes(hhex^-/-)knock out results in early death,and hhex^-/-is severely deformed at 5dpf（such as body enlargement.,small eyes,no swim bladder,small head and small liver）.Although the mutant can grow a small liver,the mutant can only survive for six days.The MTZ/NTR injury model of liver regeneration requires continuous observation of 8dpf（48 hours of regeneration）,which limits the condition to explore the cellular mechanism of liver regeneration by using hhex^-/-.Further experiments found that the heterozygote mutant(hhex^+/-)can inhibit liver regeneration,indicating that the regulation of liver regeneration by hhex may have dose-dependent.Therefore,we used hhex heterozygotes to explore the mechanism of hhex involved in liver regeneration.In the early stage of zebrafish liver regeneration,mature bile duct cells dedifferentiate into bipotent precursor cells and express hhex,sox9b and foxa3.In order to further determine whether the initiation of liver regeneration is inhibited,we used FISH-IF technique to detect and then we found that the expression of the dedifferentiation marker sox9b was down-regulated in the early stage of liver regeneration.In the process of hhex^+/-regeneration,detection of proliferation-related factors and markers found that the expression of proliferation marker genes ccnd1 and PCNA was down-regulated,and the number of Ed U-labeled cells decreased.The hhex^+/-liver was regenerated for 48 hours.Compared with the wild-type control group,the expression of hepatocytes markers gc,cp,and bhmt was significantly reduced.Therefore,hhex mainly regulates the dedifferentiation,cell proliferation and redifferentiation in the process of the liver regeneration.In order to further explore the molecular mechanism of hhex regulating liver regeneration,we preliminarily detected the important regulatory pathways involved in liver regeneration.The results showed that the expression of the marker gene tbx2b in the BMP signaling pathway was significantly down-regulated,while the expression of id2a and smad5 was slightly down-regulated;the expression of the marker genes lef1 and myca in the WNT signaling pathway was down-regulated.The results suggest that hhex may contribute to liver regeneration through BMP and WNT signaling pathways.To further confirm whether hhex regulates liver regeneration through WNT signaling or not,we overexpressed myc,an important downstream regulator of WNT,and found that myc overexpression can partially rescue the reduced liver regeneration of hhex^+/-.In conclusion,we believe that hhex affects liver regeneration by affecting cell dedifferentiation,proliferation and redifferentiation.This study further proves that the hhex gene plays an important role in the progress of liver regeneration.