The Distribution Patterns Of Four Developmental Regulators MRNA In Early Chicken Embryos And Investigate Their Roles In Developing Embryo

The sense and antisense digoxigenin-labeled RNA probes of Cdc25A, Cdc25B, Sox2 and Mnb/Dyrk four genes were produced by using SP6 and T7 RNA polymerases respectively and in vitro transcription. Expression patterns of four genes were detected by in situ hybridization in various developmental HH(Hamburger and Hamilton) stages of the early chick embryo. The results indicate that expression patterns of four genes were similar or complementary. Four genes mRNA was mostly restricted to the entire CNS (central nervous system). All of them have become confined to an identical region, neural tube, neural groove or caudal neural plate, corresponding to spinal cord. But there was some distinction in specific region or in concentration, for example in somites. Four genes mRNA was overlapping in the head region. Cdc25A and Cdc25B mRNA was complementary in CNS except head. The overlap in expression in the same developmental stage in CNS suggests that four genes may be functional similar or relation in CNS development. Expression patterns of four genes support specific roles of these regulators in the developing CNS.Experimentâ… Preparation of chicken Cdc25A RNA probe To study the distribution of Cdc25A mRNA in early chicken embryo, the sense and anti-sense digoxigenin (DIG) labeled RNA probe were prepared. The fragment of Cdc25A gene was obtained by RT-PCR through total RNA of chicken embryos. Amplified cDNA fragment was subcloned into pGM-T vector, and the plasmid was transformed into E. coli DH5a and chosen by "white-blue plaque selection". The recombinant plasmid was identified by EcoR I restriction enzyme digestion and sequencing, then Cdc25A/pGM-T vector was linearized with the restriction enzyme of Nco I and Spe I respectively. The sense and anti-sense DIG labeled RNA probe were producted by SP6 and T7 RNA polymerase respectively and transcription in vitro according to the protocol of "DIG RNA Labeling Kit (SP6/T7)".Experimentâ…¡Cdc25A mRNA was detected in chicken embryos by whole mount in situ hybridization histochemistry The distribution of Cdc25A mRNA in chicken embryo was examined by whole mount in situ hybridization histochemistry (ISHH). In stage 9HH, Cdc25A mRNA was weak in rostral head and little in the somites. But there was fruitful in the neural tube, neural groove and neural plate. In stage 10HH, Cdc25A mRNA expression was most redundant in rostral head fold. The caudal neural plate was fairly plentiful, but the middle neural tube was little. In addition, the mRNA was weak in somites. In stage 11HH, Cdc25A mRNA expression was most redundant in rostral head fold. The middle neural tube had more mRNA than prior stages. The mRNA was weak in neural groove and caudal neural plate. In addition, no Cdc25A mRNA was detected in heat primordium. The distribution of Cdc25A mRNA was in neuroepithelium.Experimentâ…¢Preparation of chicken Cdc25B RNA probe and its detection in neural tube,somites and limb bud in chicken embryos by whole mount in situ hybridization histochemistry In order to study the distributions of Cdc25B mRNA in chicken embryos, the sense and anti-sense digoxigenin (DIG) labeled RNA probes were prepared. The distributions of Cdc25B mRNA in the embryos were examined by means of whole mount in situ hybridization histochemistry (ISHH). In stage 5HH, Cdc25B mRNA was only emerged in the left of Hensen's node. In stage 6HH, Cdc25B mRNA was visible in head fold; distribution of Cdc25B mRNA was asymmetrical in Hensen's node. In stage 8-HH embryos, Cdc25B mRNA was conspicuous in the rostral head fold, and the mRNA of Cdc25B was very abundant in the neural fold; the staining of the neural fold was symmetrical, but that of Hensen's node was still asymmetrical. Cdc25B transcripts were present on both sides of the floor plate, displayed a ventro-dorsally decreasing gradient in the closing neural tube. The somites were faintly visible. In stage 8HH, the transcripts of Cdc25B were plentiful in the head fold. The somites were made clearly visible by staining. From stage 9- to 10HH, mRNA of Cdc25B was confined to the CNS, including the brain, neural tube, and neural groove. But in stage 10HH, the staining was scarce in the caudal neural plate. Cdc25B mRNA was conspicuous in the somites. In stage 12HH, the staining was stronger than in the previous stages in the CNS, especially in the head fold. Cdc25B mRNA was obvious in the somites. In stage 18HH embryos, Cdc25B was found to be still expressed in a discrete ventral domain in the neural tube. Cdc25B mRNA was detected in the tip of the limb bud. In stage 23HH embryos, Cdc25B was broadly expressed in the developing spinal cord. In general, expression patterns of Cdc25B were restricted in the CNS, somites and the tip of the limb bud in different HH stages during embryo development.Experimentâ…£Distribution of Sox2 in CNS of chicken embryo by whole mount in situ hybridization In order to study the distributions of Sox2 mRNA in chicken embryos, the sense and anti-sense digoxigenin (DIG) labeled RNA probes were prepared. The distributions of Sox2 mRNA in the stage 10HH in chicken embryos were examined by means of whole mount in situ hybridization histochemistry (ISHH). Sox2 was predominantly expressed in the immature neural epithelium of the entire CNS. Sox2 express in a band along the length of most of the CNS and in all compartments of the brain. Sox2 mRNA expression was redundant in rostral head fold according to the staining. The middle neural tube was weak of expression. The caudal neural plate was fairly plentiful. In addition, Sox2 expression was not visible in somites.In general, Sox2 mRNA was detected in the proliferative neural epithelium. The distribution of Sox2 and Cdc25A mRNA was similar in stage 10HH in chicken embryos.Experiment V Distribution of Mnb/Dyrk in CNS of chicken embryo by whole mount in situ hybridization In order to study the distributions of Mnb/Dyrk mRNA in chicken embryos, the sense and anti-sense digoxigenin (DIG) labeled RNA probes were prepared. The distributions of Mnb/Dyrk mRNA in the stage 10HH in chicken embryos were examined by means of whole mount in situ hybridization histochemistry (ISHH). Mnb/Dyrk mRNA was considerable in CNS. In HH stage 10, Mnb/Dyrk mRNA can be detected at different levels of the neural tube:the prospective prosencephalon, mesencephalon, rhombencephalon, and spinal cord. The mRNA was redundant in rostral head fold. But it was weak in caudal neural plate. Moreover, Mnb/Dyrk mRNA was very poor in somites. The distribution of Mnb/Dyrk and Cdc25B mRNA was similar in stage 10HH in chicken embryos.