Daple Gene Expression In Spatial Distribution Of Rat Brain And The Body

ObjectivesDaple is named as Dishevelled-associating protein with a high frequency of leucine residues. Daple belongs to Girdin-family genes (Girdin, Daple, FLJ004354) Among those, Daple and Girdin show considerably high homology in N-terminal domains (54%amino acids identity), which implies the functional analogy between two genes in mice, however C-terminal domains lacks similarities(only27%amino acids identity) which means Daple has its own distinctive function. Daple plays a negative control over Wnt canonical pathway, inhibiting β-catenin aggregation and Tcf transcription. By contrast, Daple has a positive impact on Wnt non-canonical pathway by enhancing PKC/Dvl interaction and activation of Rac protein. Daple is known to play an important role in postnatal cell migration. It could possibly influence fetus development, angiogenesis and tumor development. Daple’s distribution is still unknown. For the purpose of understanding related mechanisms of diseases, Daple’s function and distribution should be under study. Here we performed preliminary study to know the distribution of Daple gene expression.Materials and methodsDaple knock mice generated by SA (Splicing acceptor) trap vector techniques were purchased from TransGenic Co.,ltd. Seven-day-old (P7; female) and8-week-old mice (P56; male)(wild-type mice (+/+) and homozygous mice (-/-)) were perfused with4%PFA, then tissues were dissected, frozen, cryosectioned, and thaw-mounted on the glass slide. Daple expression was visualized using LacZ staining. To confirm LacZ staining results, in situ hybridization (ISH) was carried out using33P or Digoxigenin (DIG) labeled cRNA probe targeting Daple specific sequences (exon30). According to the distribution of Daple in the brain, we did immunohistochemistry to the brain tissue by using PGP9.5and GFAP probe. We could then determine whether Daple expresses in neuron or glial cells. We also intended to examine the possible abnormal enzyme by PCR and RT-PCR.ResultsLacZ staining of P7and P56brain exhibited significant expression of beta-geo (fusion of LacZ and Neomycin resistant gene driven by Daple promoter) in caudate putamen (striatum), ependymal cells of ventricles, cerebellum (cortex and presumably in Purkinje cells), hippocampus, olfactory bulb, bronchi, bladder, hair, tongue, stomach, intestine, spleen, spinal cord, pancrease, caridial muscle, retina, renal papillary and portal tract only in Daple (-/-) mice. ISH results confirmed the findings in LacZ staining.We found that in choroid plexus and hippocampus CA3area, daple expresses in glial cells whereas in striatum, daple expresses in both glial cells and neuron cells. Tyrosine hydroxylase (TH) RT-PCR and real time PCR results show that TH expression has no difference between homo daple knock out mice and wildtype mice.ConclusionWe succeeded in depicting the preliminary map of Daple expression. Daple exhibited significant expression in brain, spinal cord, bronchi,bladder, hair, tongue, stomach, intestine, spleenpancrease, caridial muscle, retina, renal papillary and portal tract. Daple expresses most prominently in brain is that striatum, ependymal cells of ventricles, hippocampus, dentate gyrus, substantia nigra. In choroid plexus and hippocampus CA3area, daple expresses in glial cells whereas in striatum, daple expresses in both glial cells and neuron cells. Tyrosine hydroxylase (TH) RT-PCR and real time PCR results show that TH expression has no difference between homo daple knock out mice and wildtype mice, indicating Dopamine concentration no difference between them. We hope through understanding the distribution of Daple in systematic organs and CNS, we could more clearly figure out the way that Wnt pathway works and play an fundamental basis for the future neurological function study of Daple knock out mice.