Role Of MCM5 In Hindbrain Facial Motor Neuron Development And The Underlying Mechanism

ObjectivesMinichromosome maintenance proteins5(MCM5) is one of the tiny chromosomes maintains protein family, which plays an important role in the initiation and extension of DNA replication. The expression quantity of MCM5 is far more than the amount necessarily used in DNA replication of the cell cycle, suggesting that MCM5 is still involved in other transcription function during embryo development, besides DNA replication. The early reference documents showed that MCM5 affected the growth of organisms via regulating the transcription of specific genes and participating in initiation and extension of DNA replication. In this research, we use Morpholino method to reduce the expression of MCM5, then observe the effect of the lack of MCM5 on the development of hindbrain facial motor neuron and its possible mechanism. These results contribute to understanding the molecular regulatory mechanism of the development of hindbrain facial motor neuron, and provide theoretical basis for treatment to facial motility disease caused by the hindbrain facial motor neuron developmental defects. This research also deepen our recognition of the dual role of MCM5 in cell cycle and cell differentiation.MethodsThe Morpholino was used to downregulate mcm5. The appearance of embryonic development was observed in 13, 31, 48 hpf of embryonic development by dissecting microscope. Embryonic development to 48 hpf, whole embryo islet1 antibody immunofluorescence and laser confocal microscope were used to observe the development of facial motor neurons in hindbrain. After MCM5 functional knockdown,we used the whole embryo in situ hybridization to test the expression of molecules related to FGF signaling pathway when embryonic development to 13 somites. Also, we used TUNEL staining to detect cell apoptosis at embryonic development to 48 hpf. Whole embryo pH3 antibody immunofluorescence was used to observe cell proliferative changes at 48 hpf in hindbrain. Real-time quantitative PCR(dye method)was used to detect the expression of apoptosis-related genes and proliferation-related genes. Constructing fgfr1a-pcs2 + vectors, mRNA was prepared in vitro to observewhether fgfr1 a mRNA can rescue facial motor neurons developmental defects caused by mcm5 MO in hindbrain.Results(1)The Morpholino was used to downregulate mcm5,we found that embryonic brain ventricle getting smaller, midbrain-hindbrain edge being blur. But embryonic zebrafish growth is not affected by mcm5 MO. After mcm5 functional knockdown,whole embryo islet1 antibody immunofluorescence and laser confocal microscope were used to observe the development of facial motor neurons,which found that facial motor neurons difficultly migrating in hindbrain.From co-injection of mcm5 mRNA and mcm5 MO at one cell stage of embryo, we find that mcm5 mRNA rescues difficult migration of facial motor neurons caused by mcm5 MO. MCM5 regulate development and migration of hindbrain facial motor neuron.(2) After MCM5 functional knockdown, we used the whole embryo in situ hybridization to test the expression of molecules related to FGF signaling pathway when embryonic development to 13 somites. It was found that the expression of fgf8, a ligand of FGF signaling pathway, is increased, while receptors fgfr1 a, pea3 are reduced. FGF pathway molecules expression disorder can be rescued by mcm5 mRNA.The embryo was heated shock expression dnFGFR-GFP(non-functional FGFR) for competitive inhibition FGFR activity at 80% of gastrulation outsourcing by Tg(hsp70:dnFGFR-GFP) transgenic fish.We found that FGFR activity was decreased,fgf8 activity was increased and pea3 activity was decreased,which suggested that decline of FGFR activity lead to the reverse regulation fgf8 activity was increased. The experiment confirmed that MCM5 regulated the FGF signaling activity during embryo hindbrain development,which was receptor or the further downstream in the FGF signaling pathway.(3) After mcm5 functional knockdown, TUNEL staining found that hindbrain cell apoptosis no significant changes,apoptosis-related gene expression is not statistically significant. MCM5 was not regulation cell death involved in regulation development of hindbrain neurons.(4)After MCM5 functional knockdown,wefound that decline of cell proliferation in hindbrain,proliferation-related gene expression was decreased,and mcm5 MOfunctional deficiency can be rescued by mcm5 mRNA. mcm5 regulated cell proliferation and involved in regulation development of hindbrain neurons.(5) The previous data indicated mcm5 regulation FGF signaling activity at receptor level or further downstream during embryo development. Thus,the experiment further verified whether mcm5 regulation the expression of fgfr1 a,involved in the regulation development of the facial motor neuron. The Morpholino was used to downregulate fgfr1 a, which can simulate developmental deficiency of hindbrain facial motor neurons caused by mcm5 MO. And this method can induce more serious developmental defects of facial motor neurons, it causes the facial motor neurons difficultly migrating and the number of neurons reducing. From co-injection of fgfr1 a mRNA and mcm5 MO at one cell stage of embryo, we find that fgfr1 a mRNA rescues difficult migration of facial motor neurons caused by mcm5 MO while mcm5 mRNA can’t rescue developmental deficiency of facial motor neurons after fgfr1 a functional knockdown. This experiment combined wih second part data,showed that mcm5 regulation the expression of fgfr1 a,involved in regulation development of hindbrain facial motor neurons.ConclusionsThe down-regulates the expression of MCM5 leads to embryonic brain ventricle getting smaller, midbrain-hindbrain edge being blur, facial motor neurons difficultly migrating, but it doesn’t influence brain cell apoptosis or the expression of apoptosis-related genes. However, functional knockdown of MCM5 reduces brain cell proliferation and the expression of proliferate-related genes decreases. The expression of fgf8, a ligand of FGF signaling pathway, is increased, while receptors fgfr1 a, pea3 are reduced. Specific knockdown of fgfr1 a using Morpholino can simulate developmental deficiency of hindbrain facial motor neurons caused by mcm5 MO. And this method can induce more serious developmental defects of facial motor neurons, it causes the facial motor neurons difficultly migrating and the number of neurons reducing. From co-injection of fgfr1 a mRNA and mcm5 MO at one cell stage of embryo,we find that fgfr1 a mRNA rescues difficult migration of facial motor neurons caused by mcm5 MO while mcm5 mRNA can’t rescue developmental deficiency of facial motor neurons after fgfr1 a functional knockdown. These results show that MCM5 affect the development of the facial motor neurons in hindbrain by regulating cell proliferationand adjusting fgfr1 a of FGF signaling pathway rather than by regulating apoptosis.