On the roles of Notch, Delta, kuzbanian, and inscuteable during the development of Drosophila embryonic neuroblast lineages

The generation of cellular diversity in the nervous system involves the mechanism of asymmetric cell division. Besides an array of molecules, including the Par protein cassette, a heterotrimeric G protein signalling complex, Inscuteable plays a major role in controlling asymmetric cell division, which ultimately leads to differential activation of the Notch signalling pathway and correct specification of the two daughter cells. In this context, Notch is required to be active in one sibling and inactive in the other. Here, we investigated the requirement of genes previously known to play key roles in sibling cell fate specification such as members of the Notch signalling pathway, e.g., Notch (N), Delta (Dl), and kuzbanian (kuz) and a crucial regulator of asymmetric cell division, inscuteable (insc) throughout lineage progression of 4 neuroblasts (NB1-1, MP2, NB4-2, and NB7-1). Notch-mediated cell fate specification defects were cell-autonomous and were observed in all neuroblast lineages even in cells born from late ganglion mother cells (GMC) within the lineages. We also show that Dl functions non-autonomously during NB lineage progression and clonal cells do not require Dl from within the clone. This suggests that within a NB lineage Dl is dispensable for sibling cell fate specification. Furthermore, we provide evidence that kuz is involved in sibling cell fate specification in the central nervous system. It is cell-autonomously required in the same postmitotic cells which also depend on Notch function. This indicates that KUZ is required to facilitate a functional Notch signal in the Notch-dependent cell for correct cell fate specification. Finally, we show that three neuroblast lineages (NB1-1, NB4-2, and NB7-1) require insc function for sibling cell fate specification in cells born from early GMCs whereas insc is not required in cells born from later GMCs of the same lineages. Thus, there is differential requirement for insc for cell fate specification depending on the stage of lineage progression of NBs.     

optix functions as a link between the retinal determination network and the dpp pathway to control morphogenetic furrow progression in Drosophila

optix, the Drosophila ortholog of the SIX3/6 gene family in vertebrate, encodes a homeodomain protein with a SIX protein–protein interaction domain. In vertebrates, Six3/6 genes are required for normal eye as well as brain development. However, the normal function of optix in Drosophila remains unknown due to lack of loss-of-function mutation. Previous studies suggest that optix is likely to play an important role as part of the retinal determination (RD) network. To elucidate normal optix function during retinal development, multiple null alleles for optix have been generated. Loss-of-function mutations in optix result in lethality at the pupae stage. Surprisingly, close examination of its function during eye development reveals that, unlike other members of the RD network, optix is required only for morphogenetic furrow (MF) progression, but not initiation. The mechanisms by which optix regulates MF progression is likely through regulation of signaling molecules in the furrow. Specifically, although unaffected during MF initiation, expression of dpp in the MF is dramatically reduced in optix mutant clones. In parallel, we find that optix is regulated by sine oculis and eyes absent, key members of the RD network. Furthermore, positive feedback between optix and sine oculis and eyes absent is observed, which is likely mediated through dpp signaling pathway. Together with the observation that optix expression does not depend on hh or dpp, we propose that optix functions together with hh to regulate dpp in the MF, serving as a link between the RD network and the patterning pathways controlling normal retinal development.     

Eye formation in the absence of retina

Eye development is a complex process that involves the formation of the retina and the lens, collectively called the eyeball, as well as the formation of auxiliary eye structures such as the eyelid, lacrimal gland, cornea and conjunctiva. The developmental requirements for the formation of each individual structure are only partially understood. We have shown previously that the homeobox-containing gene Rx is a key component in eye formation, as retinal structures do not develop and retina-specific gene expression is not observed in Rx-deficient mice. In addition, Rx−/− embryos do not develop any lens structure, despite the fact that Rx is not expressed in the lens. This demonstrates that during normal mammalian development, retina-specific gene expression is necessary for lens formation. In this paper we show that lens formation can be restored in Rx-deficient embryos experimentally, by the elimination of β-catenin expression in the head surface ectoderm. This suggests that β-catenin is involved in lens specification either through Wnt signaling or through its function in cell adhesion. In contrast to lens formation, we demonstrate that the development of auxiliary eye structures does not depend on retina-specific gene expression or retinal morphogenesis. These results point to the existence of two separate developmental processes involved in the formation of the eye and its associated structures. One involved in the formation of the eyeball and the second involved in the formation of the auxiliary eye structures.     

Drosophila big brain does not act as a water channel, but mediates cell adhesion

The neurogenic gene Drosophilabig brain (bib) has a high sequence homology to aquaporin-4. However, its cellular functions in Drosophila neurogenesis have remained elusive. Here we investigated cell adhesion, and the ion and water permeability of Bib. The adhesive function was examined by a cell aggregation assay using L cells. Bib-transfected L cells formed aggregated clusters, while control-L cells remained as a single cell suspension. Ion permeation was not confirmed in L cells stably expressing Bib. When expressed in COS7 cells, Bib exhibited limited water permeability. This newly found cell adhesive function of Bib may be important for Drosophila neurogenesis.     

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.     

Role of Bmbuffy in hydroxycamptothecine-induced apoptosis in BmN-SWU1 cells of the silkworm, Bombyx mori

Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells.     

Cell number counts - The fw2.2 and CNR genes and implications for controlling plant fruit and organ size

Two key determinants of plant and organ size are cell number and cell size, and altering either one may affect the plant organ size, but cell number control often plays a predominant role in natural populations. Domesticated crops usually have larger fruit and harvested organ sizes than wild progenitors. Crop yields have increased significantly by breeding, often via heterosis, which is associated with increased plant and organ size primarily achieved by cell number increases. A small class of genes is now known that control plant and organ sizes though cell number or cell size. The fw2.2 gene was found to control a major QTL for tomato fruit size by negatively affecting cell numbers. Orthologs to these fw2.2 genes underlie QTLs for fruit sizes in other species, and their expression can be negatively correlated with increased cell number. In maize decreased or increased expression of the fw2.2 ortholog ZmCNR1, increases or decreases cell number, respectively, thereby affecting maize organ size throughout the plant and thus also whole plant size. Therefore, these genes should now be considered as more general regulators of plant cell number and organ size. The exact molecular function of these transmembrane domain proteins remains unknown, as does any clear relationship to the cell cycle. Because these genes control organ sizes in diverse plants and important crop species, and because they can affect whole plant size, interest arose into how effects of such genes could parallel agronomic crop improvements, in particular that by heterosis, as it also affects cell number. In joining these subjects here in discussion we speculate on how single gene cell number regulation and heterosis may cooperate in crop improvement.     

Msx2 alters the timing of retinal ganglion cells fate commitment and differentiation

Timing of cell fate commitment determines distinct retinal cell types, which is believed to be controlled by a tightly coordinated regulatory program of proliferation, cell cycle exit and differentiation. Although homeobox protein Msx2 could induce apoptosis of optic vesicle, it is unclear whether Msx2 regulates differentiation and cell fate commitment of retinal progenitor cells (RPCs) to retinal ganglion cells (RGCs). In this study, we show that overexpression of Msx2 transiently suppressed the expression of Cyclin D1 and blocked cell proliferation. Meanwhile, overexpression of Msx2 delayed the expression of RGC-specific differentiation markers (Math5 and Brn3b), which showed that Msx2 could affect the timing of RGCs fate commitment and differentiation by delaying the timing of cell cycle exit of retinal progenitors. These results indicate Msx2 possesses dual regulatory functions in controlling cell cycle progression of retinal RPCs and timing of RGCs differentiation.     

Global/temporal gene expression analysis of Escherichia coli in the early stages of symbiotic relationship development with the cellular slime mold Dictyostelium discoideum

Escherichia coli and the cellular slime mold Dictyostelium discoideum form stable viscous symbiotic colonies in the laboratory. To examine changes in E. coli gene expression during establishment of this symbiotic relationship, cells of symbiotic co-cultures and monocultures at various time points were subjected to microarrays analysis. Genes changed significantly over time compared to the initial gene expression level were determined as characteristics of GO function categories. The categories that appeared significantly at the same sampling time points between the two cultures were also identified. Up-regulation of genes from several GO categories associated with polysaccharide synthesis, cell wall degradation, and iron acquisition as well as down-regulation of genes from GO categories associated with biosynthesis through starvation response were observed in co-cultures, indicating exchange of molecules between the two organisms. Up-regulation of genes from several GO categories associated with anaerobic respiration and flagella biosynthesis were also observed, indicating that the environment inside symbiotic colonies was similar to that in developed biofilms. Up-regulation of genes associated with energy-generating systems indicated that E. coli prolonged survival within the symbiotic colony. Thus, E. coli showed not only molecule exchange but also altered expression of various genes in symbiosis with D. discoideum.     

Bestrophin genes are expressed in Xenopus development

The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.