In situ hybridization identifies sites of Drosophila AKH synthesis towards the base of the third larval instar ring gland. Like other RPCH (red pigment concentrating hormone)/AKH family peptides, DAKH can act as a cardioaccelerator at least in prepupae. Peptide levels measured in wildtype and mutant flies possessing one or three copies of the DAKH gene suggest that the amount of neuropeptide per fly is tightly regulated (Noyes, 1995).

The larval ring gland is an important endocrine organ in the cyclorraphous Diptera, consisting of the corpus allatum (CA), prothoracic gland, and corpora cardiaca (CC). The dAkh gene was cloned and shown to produce mRNA exclusively in the CC of third-instar larvae (Noyes, 1995); however, dAkh-expressing tissue types beyond this juvenile stage have not been examined (Lee, 2004).

Prior to the examination of dAkh expression patterns in adult flies, whole-mount in situ hybridizations was performed on third-instar larval tissues to validate a new antisense dAkh riboprobe. In agreement with previous results, the probe produced specific signals exclusively in the CC. Using this probe, the assay was extended to adult tissues. During metamorphosis, the ring gland migrates posteriorly and finally attaches to the esophagus, just anterior to the cardia (or proventriculus) in the adult thorax. Strong and unique in situ hybridization signals were detected in a tiny structure located at this position. The results suggest that the CC-specific dAkh expression pattern remains unchanged during metamorphosis (Lee, 2004).

To determine whether AKH peptides are actually synthesized in the CC cells, whole-mount immunohistochemistry was performed using anti-AKH antibodies. Consistent with dAkh mRNA expression patterns, AKH-immunoreactive signals were limited to the CC of both larvae and adults, suggesting that intrinsic neurosecretory cells in the CC actively produce AKH peptides during both juvenile and adult stages. Essentially identical expression patterns obtained by both techniques also verify the specificity of the antibody to the AKH peptides. From the results, it is concluded that the CC is the only tissue type expressing the dAkh gene in Drosophila melanogaster (Lee, 2004).

Using the Gal4-UAS system to drive lacZ in the pattern of normal Akh expression (dAkh-gal4/UAS-lacZ), the earliest developmental stage of dAkh expression was determined. The ß-gal activity was at first faint in a paired structure in approximately stage-14 embryos and then became stronger in older embryos. CC-specific expression was also observed in first-instar larvae; however, projections from the CC neurons were undetectable, suggesting that the dAkh neurons in first-instar larvae have not yet been fully differentiated. Nevertheless, the overall results suggest that normal dAkh gene functions might be necessary from late embryonic stages onward (Lee, 2004).

Little is known about neuro-anatomical details of the intrinsic neurosecretory cells in the CC of Drosophila. Since dAkh gene products could serve as a useful marker for such cells, characteristics of these cells were further examined in great detail, using various transgenic manipulations and histochemical assays. (1) In determining the number of dAkh-expressing cells, dAkh-gal4 flies were crossed to a UAS-NZ reporter to express ß-gal in the nuclei of dAkh cells. As a result, ~7 cells per each lobe of larval CC were identified. For adult CC, the total number of dAkh cells was counted from a whole CC structure instead of per each lobe, since the boundary between lobes was not clearly recognizable, thus hampering precise counting. This yielded an average of 13 cells per CC, ranging from 11 to 16. Since the counts in an adult CC are comparable to those observed in an entire larval CC, dAkh cells might be present persistently during metamorphosis (Lee, 2004).

To determine the population of dAkh cells in the CC, somata of dAkh neurons were simultaneously marked by dAkh promoter-driven gfp expression and nuclei of entire CC cells were marked DAPI staining. A majority of the DAPI-positive cells expressed gfp, suggesting that dAkh cells represent most of the CC cells (Lee, 2004).

Stainings mediated by anti-AKH antibody and X-gal histochemistry were examined at higher resolution to construct a fine neural mapping involving the AKHergic neurons. In larvae, two potential targets were detected innervated by AKHergic neurons, one of which is the prothoracic gland located immediately adjacent to the CC and known to produce a molting hormone ecdysteroid. The AKHergic neurons sent two or three projections to this structure. The other target is the aorta (or dorsal vessel) that is closely associated with the CC. Extensive AKH-immunoreactive varicosities observed on the aorta indicate that AKHs are released into the circulatory system (Lee, 2004).

Although it is not as clear as in larval CC, adult CC also form a bi-lobed structure and the dAkh neurons are present in both lobes. Processes stemming from the anterior side of the lobes were traced proximate to the esophagus foramen where they are likely to enter the protocerebrum. A pair of long processes arising from the posterior side reached the crop duct at which the crop begins its expansion. In some insects, such as honeybees and blow flies, the crop stores liquid foods (e.g., nectar or soluble nutrients), and its volume is highly variable depending on the amounts of liquid deposit. AKH-homologous peptides have been proposed to cause regurgitation of nectars from the crop in some wasp species to increase hemolymph trehalose titers. In this regard, the findings of AKH nerve terminals at the crop duct support the idea that AKH may control the crop volume in Drosophila. In addition to the brain and the crop, a process whose target could not be identified was occasionally observed. Nonetheless, this implies additional physiological functions attributed to AKH in adult flies (Lee, 2004).

Reference names in red indicate recommended papers.

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Park, Y., Kim, Y. J. and Adams, M. E. (2002). Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution. Proc. Natl. Acad. Sci. 99: 11423-11428. 12177421

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date revised: 10 December 2004

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