As interconnected spermatocytes begin to grow, there is a substatial upregulation of orb2 mRNA and protein. orb2 expression peaks as the mature spermatocytes go through meiosis and high levels of orb2 are found in 32 and 64 cell spermatid cysts. In elongating spermatids, orb2 is distributed along the entire axoneme bundle. The highest concentrations are found in a prominent band close to the distal tip of the growing flagellar axonemes. In the region behind the band, orb2 is organized into a series of striated lines that extend towards the sperm nuclei at the proximal (basal) tip of the spermatid and presumably correspond to individual flagellar

axonemes in the spermatid bundle. orb2 disappears once elongation and nuclear condensation are completed.

orb2 protein is deposited maternally and expressed throughout embryogenesis with a decrease in expression between 4-8h AEL. Expression of orb2 protein is detected in the cell bodies of neurons in the CNS and PNS, being excluded from longitudinal and commissural axon tracts. orb2 protein labels the lineage of the Even-skipped positive neuroblast NB4-2. It also labels the cytoplasm of mesodermal cells, in which it co-localises with ScerGAL4^twi.PU. Western blot analysis indicates that in adults, the isoform orb2-PA is restricted to the testis, while the other isoforms orb2-PB,PC and PD are detected in ovaries and testis. orb2 protein is detected in the cell bodies of a subset of neurons in the larval brain and ventral nerve cord. In the adult, orb2 protein is expressed in the ellipsoid body, in two unindentified and symmetrical structures dorsal to the ellipsoid body, in an unpaired structure on the left hemisphere, lateral to the ellipsoid body and in the fan-shaped body. In adult neurons, orb2 protein seems to be localised to the synaptic terminals. In the ovaries, in previtellogenic stage chambers, orb2 protein is detected in all of the cells but enriched in a cap posterior to the oocyte. At oogenesis stages S8 to S10, orb2 protein is enriched in the cortex of the oocyte, similarly to orb protein. orb2 protein is expressed around the nucleus of the nurse cells, unlike the orb2 transcript. It is also detected in a gradient in the cytoplasm of the nurse cells, with expression being strongest in the most posterior cells (closest to the oocyte). orb2 protein is released from these cells into the oocyte via ring canals.