Multiple isoforms of the Activin-like receptor baboon differentially regulate proliferation and conversion behaviors of neuroblasts and neuroepithelial cells in the Drosophila larval brain.

In Drosophila coordinated proliferation of two neural stem cells, neuroblasts (NB) and neuroepithelial (NE) cells, is pivotal for proper larval brain growth that ultimately determines the final size and performance of an adult brain. The larval brain growth displays two phases based on behaviors of NB and NEs: the first one in early larval stages, influenced by nutritional status and the second one in the last larval stage, promoted by ecdysone signaling after critical weight checkpoint. Mutations of the baboon (babo) gene that produces three isoforms (BaboA-C), all acting as type-I receptors of Activin-type transforming growth factor ß (TGF-ß) signaling, cause a small brain phenotype due to severely reduced proliferation of the neural stem cells. In this study we show that loss of babo function severely affects proliferation of NBs and NEs as well as conversion of NEs from both phases. By analyzing babo-null and newly generated isoform-specific mutants by CRISPR mutagenesis as well as isoform-specific RNAi knockdowns in a cell- and stage-specific manner, our data support differential contributions of the isoforms for these cellular events with BaboA playing the major role. Stage-specific expression of EcR-B1 in the brain is also regulated primarily by BaboA along with function of the other isoforms. Blocking EcR function in both neural stem cells results in a small brain phenotype that is more severe than baboA-knockdown alone. In summary, our study proposes that the Babo-mediated signaling promotes proper behaviors of the neural stem cells in both phases and achieves this by acting upstream of EcR-B1 expression in the second phase.

In vivo characterization of the maturation steps of a pigment dispersing factor neuropeptide precursor in the Drosophila circadian pacemaker neurons.

Pigment dispersing factor (PDF) is a key signaling molecule coordinating the neuronal network associated with the circadian rhythms in Drosophila. The precursor (proPDF) of the mature PDF (mPDF) consists of 2 motifs, a larger PDF-associated peptide (PAP) and PDF. Through cleavage and amidation, the proPDF is predicted to produce cleaved-PAP (cPAP) and mPDF. To delve into the in vivo mechanisms underlying proPDF maturation, we generated various mutations that eliminate putative processing sites and then analyzed the effect of each mutation on the production of cPAP and mPDF by 4 different antibodies in both ectopic and endogenous conditions. We also assessed the knockdown effects of processing enzymes on the proPDF maturation. At the functional level, circadian phenotypes were measured for all mutants and knockdown lines. As results, we confirm the roles of key enzymes and their target residues: Amontillado (Amon) for the cleavage at the consensus dibasic KR site, Silver (Svr) for the removal of C-terminal basic residues from the intermediates, PAP-KR and PDF-GK, derived from proPDF, and PHM (peptidylglycine-α-hydroxylating monooxygenase) for the amidation of PDF. Our results suggest that the C-terminal amidation occurs independently of proPDF cleavage. Moreover, the PAP domain is important for the proPDF trafficking into the secretory vesicles and a close association between cPAP and mPDF following cleavage seems required for their stability within the vesicles. These studies highlight the biological significance of individual processing steps and the roles of the PAP for the stability and function of mPDF which is essential for the circadian clockworks.

Regulation of ethanol-related behavior and ethanol metabolism by the Corazonin neurons and Corazonin receptor in Drosophila melanogaster.

Impaired ethanol metabolism can lead to various alcohol-related health problems. Key enzymes in ethanol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH); however, neuroendocrine pathways that regulate the activities of these enzymes are largely unexplored. Here we identified a neuroendocrine system involving Corazonin (Crz) neuropeptide and its receptor (CrzR) as important physiological regulators of ethanol metabolism in Drosophila. Crz-cell deficient (Crz-CD) flies displayed significantly delayed recovery from ethanol-induced sedation that we refer to as hangover-like phenotype. Newly generated mutant lacking Crz Receptor (CrzR(01) ) and CrzR-knockdown flies showed even more severe hangover-like phenotype, which is causally associated with fast accumulation of acetaldehyde in the CrzR(01) mutant following ethanol exposure. Higher levels of acetaldehyde are likely due to 30% reduced ALDH activity in the mutants. Moreover, increased ADH activity was found in the CrzR(01) mutant, but not in the Crz-CD flies. Quantitative RT-PCR revealed transcriptional upregulation of Adh gene in the CrzR(01) . Transgenic inhibition of cyclic AMP-dependent protein kinase (PKA) also results in significantly increased ADH activity and Adh mRNA levels, indicating PKA-dependent transcriptional regulation of Adh by CrzR. Furthermore, inhibition of PKA or cAMP response element binding protein (CREB) in CrzR cells leads to comparable hangover-like phenotype to the CrzR(01) mutant. These findings suggest that CrzR-associated signaling pathway is critical for ethanol detoxification via Crz-dependent regulation of ALDH activity and Crz-independent transcriptional regulation of ADH. Our study provides new insights into the neuroendocrine-associated ethanol-related behavior and metabolism.

Mechanisms of postecdysis-associated programmed cell death of peptidergic neurons in Drosophila melanogaster.

Crustacean cardioactive peptide (CCAP)-expressing neurons undergo programmed cell death (PCD) within 24 hours after adult eclosion. A subset of the doomed CCAP neurons in the ventral nerve cord also expressed the neuropeptide bursicon and thus are referred to as bursCCAP neurons. In this study, we undertook comprehensive genetic and transgenic analyses to dissect the PCD mechanisms of bursCCAP neurons. Expression of a versatile caspase inhibitor, p35, blocked PCD of bursCCAP neurons, suggesting caspase-dependent apoptosis. Further genetic analyses showed that Dronc/Dark and Drice are key caspases, but they are not sufficient to carry out the PCD fully. We did not find a role for other known caspases, Strica, Dredd, Damm, or Decay. Of interest, Dcp-1 is required not for the death of bursCCAP neurons per se but for the removal of neural projections. DIAP1 is an important survival factor that inhibits premature death of bursCCAP neurons. We found that grim functions as a principal death inducer, whereas other death genes, hid, reaper, and sickle, show no endogenous function. Taken together with other studies, our work supports the role of grim as a major death inducer particularly for the removal of obsolete larval neurons during CNS metamorphosis. Results from the ectopic expression of the mutant grim lacking either N-terminal IBM or internal GH3 domain indicated that both domains are necessary to induce CCAP cell death.