Improvement Of Paraffin Section Of Drosophila Melanogaster And Expression Of CAMR1 / 2 In Early Embryo Development Of Drosophila

Drosophila melanogaster is one of the most important model organisms with many unique advantages in developmental biology. However, difficulties in fixation process of Drosophila embryo when producing paraffin sections are also recognized meanwhile for years, which result in failure of embedding and sectioning, and hinder subsequent experiment. CAMRs regulate many important physiological progresses, but their expression and distribution within early embryonic development are infrequently researched.In this study, Drosophila melanogaster embryos were used as an experimental model combining with antibody specific immunoreaction on the sections to illustrate function of CAMRs. Firstly we modified the traditional paraffin section approach and optimized a protocol for Drosophila embryo. Secondly, we compared the effect of different embedding co-adjuvant (gelatin, agarose and paraffin) with haematoxylin eosin (HE) staining and Periodic acid-silver metheramine (PASM)staining. Histological structures of embryos in different developmental stages were displayed by HE staining meanwhile, which further supported the selection of suitable co-adjuvant for Drosophila embryo paraffin section. Moreover, with the modified method, we localized the expression of homologous genes CAMR1 and CAMR2 within Drosophila embryo early development. CAMR1 and CAMR2 showed obvious difference in distribution of expression, which indicate that these two genes have begun to function differently in Drosophila early development. Our results are as the follows:(1) Permeabilization modified paraffin gathering method is more propitious for production of high fat Drosophila embryo paraffin sectionsHE histological analysis showed that sections of unpermeabilized groups existed cavities and cracks in embryo, while permeabilized group kept good embryo morphological structure. It indicates that permeabilization plays an important role in fixative penetrating to Drosophila embryo. In addition, it also showed that sections with agarose and paraffin gathering method all displayed clear embryo lineament, integrated morphological structure, well internal staining and clean background. The result indicates that agarose and paraffin all have promising structure stability and permeability without causing obvious extrusion to embryo in process of dehydration and embedding. PASM histological analysis indicated that, compared with other two gathering methods, sections with paraffin gathering method showed uncontaminated background, clear lineament and integrated structure. Therefore, it is convinced that both agarose and paraffin gathering method perform courteously in preserving Drosophila embryo structure, and both can be applied in embedding scrawled embryo samples for subsequent experiments.In this experiment, we completed the paraffin section production of Drosophila embryos with the modified paraffin gathering method, and we carried out a series of Drosophila embryo histological analysis with HE staining and tracked the whole embryo development process in four stages (stage 1, syncytial blastoderm; stage2, Cellular blastoderm; stage3, pre-gastrulation and stage4, gastrulation). Furthermore, it established the foundation for subsequent experiments such as immunohistochemical localization of target protein.(2) Function differentiation of CAMR1 and CAMR2 in regulating Drosophila embryo developmentFirstly, with RT-PCR and Western Blot, we detected that both CAMR1 and CAMR2 genes were expressed in Drosophila early embryo displaying in mRNA level and protein level. Then, we compared the expression pattern alteration of CAMR1 and CAMR2 with immunohistochemical staining. Our results showed that, the expression of CAMR1 distributed widely in Drosophila embryogenesis, and in the progress of cellularization, its expression level was higher in blastoderm cell. Along with the progress of gastrulation, CAMR1 was highly expressed respectively in peripheral blastoderm cells with actively division, anterior ventral part of ectoderm and highly differentiated portion such as neuron system, midgut, cuticula and muscle. All above indicate that CAMR1 plays an important role in Drosophila embryogenesis and differentiation. CAMR1, which involves in regulating Ca2+ signaling, TGF-β signaling and cell cycle, may participate in regulating function differentiation of Drosophila embryogenesis, and it may be closely related to organ development such as brain and nervous system.The expression pattern alteration of CAMR2 in Drosophila early embryogenesis is obviously different from CAMR1. CAMR2 was barely expressed at syncytial stage. At the end of cellularization, it was mainly expressed in the basal layer of blastoderm, which presented evident polarity differentiation. The difference distribution of expression with highest in head and weakest in tail is extremely obvious, and in the frontal section, CAMR2 expression showed distribution characteristics of bilateral symmetry and enhanced expression level gradually from head to tail. While CAMR2 expression nearly couldn’t be detected in blastoderm cells and yolk at this stage. At gastrula stage, the dorsal and ventral parts have showed obvious difference. There were no CAMR2 expression in the dorsal monolayer epithelial cells and ventral multilayer epithelial cells, but higher expression in differentiated organs in embryo. With morphogenesis of organs, the expression of CAMR2 also displayed differential distribution. Our data demonstrated that CAMR2 participated in functional differentiation of Drosophila embryogenesis, and may performe in different ways with CAMR1.