The v-ATPase is a fundamental eukaryotic enzyme that is central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well documented, our knowledge of mechanisms that regulate v-ATPase activity is limited. Here, we report that the Drosophila transcription factor Mitf is a master regulator of this holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation affects holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. We provide evidence that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF family proteins in fly and mammals. Collectively, this evidence points to an ancient module comprising Mitf, v-ATPase and TORC1 that serves as a dynamic modulator of metabolism for cellular homeostasis.

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. We show here that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc epithelium at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. We also show that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. Our results support a critical role for Yki- and its partners Sd and Hth - in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival.

Proneural transcription factors drive the generation of specialized neurons during nervous system development, and their dynamic expression pattern is critical to their function. The activation of the proneural gene atonal (ato) in the Drosophila eye disc epithelium represents a critical step in the transition from retinal progenitor cell to developing photoreceptor neuron. We show here that the onset of ato transcription depends on two distant enhancers that function differently in subsets of retinal progenitor cells. A detailed analysis of the crosstalk between these enhancers identifies a critical role for three binding sites for the Retinal Determination factors Eyeless (Ey) and Sine oculis (So). We show how these sites interact to induce ato expression in distinct regions of the eye field and confirm them to be occupied by endogenous Ey and So proteins in vivo. Our study suggests that Ey and So operate differently through the same 3′ cis-regulatory sites in distinct populations of retinal progenitors.

Background: The major royal jelly proteins/yellow (MRJP/YELLOW) family possesses several physiological and chemical functions in the development of Apis mellifera and Drosophila melanogaster. Each protein of the family has a conserved domain named MRJP. However, there is no report of MRJP/YELLOW family proteins in the Lepidoptera.

The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. We have identified Protein Phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. We show that these factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hpo kinase, and consequently its substrate Wts, to release the transcriptional co-activator Yki into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate.

The adult fly has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2 kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter organs, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, our findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye.

The scaleless wings mutant in Bombyx mori (scaleless, sl) was previously reported morphologically. In the present study, we give data to clarify the mechanism of the mutation at the developmental level. Programmed cell death participates in the wing scale development during early pupal stage, and there are significant differences between that of sl and the wild type (WT) at each phase. Well-differentiated scale precursor cells do not form in sl when they have formed in WT. The peak of Caspase-3/7 activity in sl occurs 1 day later than and ten times as much as that in WT. Apoptotic bodies and DNA ladder studies also show that there is excessive apoptosis in sl early pupal wing. In addition, we have studied Bm-ASH1, an achaete-scute homolog in B.mori, which is thought to play a key role during the development of wing scales, and have found that the gene structure and expression levels of Bm-ASH1 in sl and WT are identical. All the data indicate that the wing scale precursor differentiation mechanism is abnormal in sl, which causes failing determination of scale cells and the downstream symptom of excessive apoptosis. But some of the elements to the scale differentiation circuit, such as Bm-ASH1, still operate in sl.

The proneural genes are fundamental regulators of neuronal development in all metazoans. A critical role of the fly proneural factor Atonal (AtoDm) is to induce photoreceptor neuron formation in Drosophila, whereas its murine homolog, Atonal7Mm (aka Ath5) is essential for the development of the ganglion cells of the vertebrate eye. Here, we identify the Bombyx mori ato homolog (atoBm). In a pattern strikingly reminiscent of atoDm, the atoBm mRNA is expressed as a stripe in the silkworm eye disc. Its DNA-binding and protein-protein interaction domain is highly homologous to the AtoDm bHLH. Targeted expression of AtoBm in the endogenous atoDm pattern rescues the eyeless phenotype of the fly ato1 mutant and its ectopic expression induces similar gain-of-function phenotypes as AtoDm. Rescue experiments with chimeric proteins show that the non-bHLH portion of AtoBm (N-region) can effectively substitute for the corresponding region of the fly transcription factor, even though no apparent conservation can be found at the amino acid level. On the contrary, the highly similar bHLH domain of AtoBm cannot similarly substitute for the corresponding region of AtoDm. Thus, the bHLHBm domain requires the AtoBm N-region to function effectively, whereas the bHLHDm domain can operate well with either N-region. These findings suggest a role for the non-bHLH portion of Ato proteins in modulating the function of the bHLH domain in eye neurogenesis and implicate specific aa residues of the bHLH in this process.