The Study On Enu Chemically Mutagenic Zebrafish Mutants Of Phosphoinositide Phosphatase Gene Fig4a And Myosin Heavy Chain Gene Myhz2

Zebrafish is an important model organism used for the study on vertebrate embryonic development and gene function,which shares 87%homologous similarity of genome sequences with human.With the features of small size,easy breeding,in vitro development,and transparent body at embryonic and early larval stages,zebrafish is an appropriate tool for mass screening work.Chemical mutagenesis with Enu,treating adult zebrafish to generate mutant offspring and identifying the lines with target phenotypes,has become one of the most common methods for studying embryonic development and gene function.During the large-scale Enu screening process for zebrafish mutants carried out in our lab,we obtained kinds of lines possessing hepatic or certain specific phenotype.In the two parts of this thesis,we will introduce the research contents about cq35 and dtb zebrafish mutants,which respectively perform severe vacuolization hepatopathy and specific tail shortening phenotype.PI（3,5）P₂ signaling pathway is an essential regulatory approach to ensure intracellular membrane traffick and lysosome-related action processes.The phospholipid molecule exhibits high efficiency in cellular physiological regulation despite of its low intracellular concentration.It has been known that PI（3,5）P₂ is synthesized by the phosphokinase PIKFYVE in PAS complex,and this kinase activity relies on the structural auxiliary roles of other complex members including FIG4.Till now,lots of research reports have demonstrated the association between FIG4 mutations and certain human diseases,especially many neurological disorders e.g.CMT4J,YVS,ALS,PLS,and familial epilepsy syndrome.Abnormal intracellular accumulation of lysosomal components and tissue degeneration usually occur in the lesion regions of patients,which are mainly focused in nerve system and musculoskeletal system as reported currently.Liver,an essential organ performing multiple functions correlated with organismal metabolism and regulation,possesses abundant lysosomal pathways in hepatocyte population to ensure its high-level metabolism and homeostasis maintenance.However,so far,no reports have correlated FIG4 gene function or mutation with liver physiology.Via Enu mutagenesis and screening,we obtain a recessive zebrafish mutant cq35exhibiting severe liver vacuolization phenotype,with the genetic mutation site localized in fig4a gene sequence.The premature termination codon owing to base substitution mutation theoretically leads to truncation of Fig4a protein,thus influencing the structural stability of PAS complex,which eventually disturbs the PI（3,5）P₂ synthesis capacity of the latter.Although some vacuole-like microstructures appear as well in certain other locations including central nerve system region,the vacuolation degree of mutant liver is preponderantly evident.The observation under Tg（lfabp:GFP）transgenic fluorescence background find that all the hepatocytes of mutant become vacuolated as time passes,and the liver gradually swelling and lose its normal shape.Meanwhile,significant individual differences exist among mutant larvae,on the aspects of onset time and pathological degree of liver vacuolation phenotype,as well as shortened life due to mutational effects.Utilizing heat shock promoter hsp70l,we construct the transgenic zebrafish line that can time-selectively express normal fig4a transcripts,in which,the heat shock treatment after fig4a^cq35 liver becomes obviously vacuolated rescues the pathological degree of liver phenotype effectively.This result not only confirms the corresponding relationship between cq35 mutant and fig4a gene,but also demonstrates the reversibility of cytosolic vacuolation phenotype caused by fig4a gene mutation.Intravital lysotracker red staining indicates that those huge vacuoles in fig4a^cq35 hepatocytes are abnormal enlarged lysosomes,containing large numbers of lysotracker-positive materials,which are also evidently labeled by Lc3 antibody in the co-staining experiment,suggesting that the autophagy process in fig4a^cq35 hepatocytes is retarded at substrate degradation step as a result of lysosomal abnormalities.Liver structure and function are influenced by vacuolation hepatopathy in fig4a^cq35 larvae,for instance,Abcb11 antibody staining shows that the bile canaliculi in mutant liver degenerate progressively as the hepatic vacuolation deteriorates,pointing to the decline or even loss of bilification function in fig4a^cq35 liver.Also,in the zebrafish mutant cq92 of pikfyve gene identified coincidently in our Enu screening work,the same vacuolization hepatopathy and related pathological details are detected,which conforms to the cooperative relationship between FIG4 and PIKFYVE in PI（3,5）P₂ synthesis function of PAS complex.Yet,the mutational effects shown in pikfyve^cq92 larvae are notably stronger than those in fig4a^cq35 larvae,and pikfyve^cq92phenotype covers fig4a^cq35 phenotype in their double mutant larvae,which can be explained by the auxiliary role of FIG4 to ensure PIKFYVE kinase activity in PAS complex,namely,fig4a^cq35 phenotype represents a kind of weakened pikfyve^cq92phenotype.Thus,zebrafish possess conservative structural and functional mechanisms of PAS complex,based on which,zebrafish provide a novel organism carrier for the research on PI（3,5）P₂ signaling pathway effects and associated diseases due to mutations of complex members including FIG4.Meanwhile,fig4a^cq35 and pikfyve^cq92 zebrafish larvae may be favorable tools used for drug screening against PI（3,5）P₂ deficiency-related diseases.In conclusion,our data firstly demonstrate the importance of FIG4/Fig4 for liver physiology,and suggest that FIG4 may be a potential pathogenic gene of certain etiology-unknown liver diseases with lysosomal storage pathology.During Enu mutagenesis and screening,we also obtain a dominant zebrafish mutant dtb with severe but specific tail shortening phenotype.Considering the evolutionary homology among vertebrates,we attempt to associate dtb phenotype with the tail degeneration of some vertebrate species or the tail disappearance of human during evolution process,which is also our interest and purpose to study this mutant.Via gene mapping,the mutational site is localized in fast muscle-specific myosin heavy chain gene myhz2,concretely in the region coding conservative amino acid sequence of ATP binding domain that locates in the head of myosin heavy chain.According to previous reports,the mutations of this specific sequence region in myosin heavy chain proteins can cause the failure of sarcomere assembly in muscle fibers,and indeed,the muscle tissue-specific transverse striation feature is not seen in caudal fast muscles of dtb embryo.In situ hybridization results show that myhz2 gene has specific expression pattern in caudal region of zebrafish embryo.Under Tg（ras GFP）transgenic fluorescence background,it can be dynamically observed that the caudal fast muscle fibers of dtb embryo gradually lose the regular fibrous shape after their formation via fusion,and the arrangement of fast muscle cells in the whole tail region becomes seriously disordered,which may be caused by the sarcomere assembly failure in those fast muscle cells.Various irregular cavity structures and prominent aggregation of inflammatory cells,such as macrophages and granulocytes,appear in the caudal fast muscle tissues of dtb embryos,where nevertheless no obvious up-regulation of apoptosis is detected,suggesting that the pathological caudal fast muscle fibers are deathward to necrosis.Suffering from the lesion and atrophy of fast muscle tissues,the developing tail spine is axially squeezed and folded to adapt to the seriously compressed tissue space in caudal region of dtb larvae,and the neural and haemal arches of caudal vertebrae are morphologically abnormal as well.Based on those phenotypic details,it can be concluded that the specific tail shortening phenotype of dtb mutant results from structural and functional abnormalities of caudal fast muscle tissues.Since the expression of myhz2 gene has significant tissue specificity and the protein encoded is a kind of intracellular protein playing structural function,the mutational effects are predominantly focused in fast muscle tissues of mutant tail,which however distinctly differs from the developmental phenomenon of human embryo that almost all of tissue types in tail region eventually disappear via necrosis.Therefore,from the aspects of gene function and phenotypic characteristics,we rule out the possibility of association between myhz2 gene and human tail disappearance.Yet,the similarities between both tail regression processes may bring us some revelations.For instance,the elimination of human embryonic tail tissues is also implemented by necrosis,a passive way of cell death,which may support the genetic mutation inference explaining human tail disappearance,namely meaning that tail loss is not an intention of human embryonic development direction.