Utilizing this model technique, we undertook studies to take a look at mechanistically the interaction among mutationally activated ALK and MYCN overexpression while in neuroblastoma pathogenesis during the PSNS. Final results Transgenic EGFP Expression in the PSNS We 1st isolated a . kb promoter fragment upstream of the coding sequence from the zebrafish dopamine b hydroxylase gene , which encodes the charge limiting enzyme for noradrenalin synthesis. This fragment was utilized to drive expression of enhanced green fluorescent protein inside a stable zebrafish transgenic line, Tg , designated DbH in this post. In juvenile and grownup transgenic zebrafish, EGFP was specifically expressed by sympathetic neurons on the superior cervical ganglia , the 1st sympathetic ganglion to produce in early embryogenesis, and by every single sequential segmental ganglion within the sympathetic chain . EGFP was also expressed by sympathoadrenal cells from the interrenal gland , the zebrafish equivalent on the human adrenal gland . During the interrenal gland, the EGFP expressing cells is usually visualized inside a discrete region within the ventral factor within the head kidney, intermixed with adrenal cortical cells which can be TH and EGFPnegative .
The specificity of EGFP expression for sympathoadrenal cells when Proteasome Inhibitor selleck driven through the dbh promoter fragment is demonstrated by coexpression of endogenous TH , an alternative enzyme expressed by sympathetic neurons and chromaffin cells . Zebrafish Expressing MYCN Develop Neuroblastoma Utilizing a coinjection approach , we created a secure transgenic zebrafish line, Tg , designated MYCN in this post, that overexpresses the human MYCN gene fused to EGFP underneath control in the dbh promoter. In MYCN transgenic fish the growth of cells expressing EGFP as tumors developed was readily detectable in living fish by immunofluorescence microscopy . EGFP tumor masses were observed from the anterior abdomen, corresponding on the interrenal gland, and had been composed of small, undifferentiated, round tumor cells with hyperchromatic nuclei, typically forming nests .
Tumor cells were strongly immunoreactive for TH as well as pan neuronal markers Hu and Synaptophysin , indicating their PSNS connected neuronal origin . Standard interrenal chromaffin cells also expressed TH, but not Hu or Synaptophysin , indicating that the neuroblastomas arose from sympathetic neuroblast precursors and never chromaffin cells, as is definitely the situation in human neuroblastoma . Neuroblastoma is commonly viewed as Procaine from the differential diagnosis of malignant little round cell tumors of childhood, and electron microscopy is actually a valuable device for distinguishing among these malignancies. A diagnosis of neuroblastoma is usually established ultrastructurally by demonstrating the presence of neurosecretory granules within the cytoplasm or cytoplasmic processes of tumor cells .