The nuclear transport machinery in Caenorhabditis elegans: A central role in morphogenesis

Our full understanding of the various roles for the nuclear transport machinery has come from a variety of model organisms including yeast, nematodes, fruit flies and vertebrates. Using the nematode Caenorhabditis elegans, it has been shown that the karyopherin family of nuclear transporters and the components of the Ran cycle have roles not only in nuclear protein transport, but also in mitotic spindle formation and regulation, and in nuclear envelope assembly. These studies have also demonstrated a role for nuclear transport factors in cellular differentiation and development, particularly for the formation of germ cells. This review highlights the small number of studies in C. elegans that have been critical to our understanding of this important cellular process.     

The Drosophila homolog of NTF-2, the nuclear transport factor-2, is essential for immune response

Nuclear transport factor-2 (NTF-2) functions in yeast and mammalian cell culture in targeting proteins into the nucleus. The Drosophila homolog, DNTF-2, is an essential component of the nuclear import machinery, since ntf mutants are lethal. Interestingly, hypomorphic alleles show specific phenotypes. Some are viable, but the number of omatidia in the eye is severely reduced. The immune response in the Drosophila larval fat body is also affected; the three NF-κB/Rel proteins Dorsal, Dif and Relish do not target to the nucleus after infection, and, consequently, the expression of the anti-microbial peptide genes drosomycin, attacin and drosocin is severely impaired. Hence, in spite of its general requirement in many developmental processes, DNTF-2 has a higher specific requirement in the development of the eye and in the immune response. We also found that DNTF-2 interacts directly with Mbo/DNup88, which does not contain phenylalanine-glycine-rich repeats, but has been shown to function in the import of Rel proteins.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

ALS2 regulates endosomal trafficking,postsynaptic development,and neuronal survival

Mutations in the human ALS2 gene cause recessive juvenile-onset amyotrophic lateral sclerosis and related motor neuron diseases. Although the ALS2 protein has been identified as a guanine-nucleotide exchange factor for the small GTPase Rab5, its physiological roles remain largely unknown. Here, we demonstrate that the Drosophila homologue of ALS2 (dALS2) promotes postsynaptic development by activating the Frizzled nuclear import (FNI) pathway. dALS2 loss causes structural defects in the postsynaptic subsynaptic reticulum (SSR), recapitulating the phenotypes observed in FNI pathway mutants. Consistently, these developmental phenotypes are rescued by postsynaptic expression of the signaling-competent C-terminal fragment of Drosophila Frizzled-2 (dFz2). We further demonstrate that dALS2 directs early to late endosome trafficking and that the dFz2 C terminus is cleaved in late endosomes. Finally, dALS2 loss causes age-dependent progressive defects resembling ALS, including locomotor impairment and brain neurodegeneration, independently of the FNI pathway. These findings establish novel regulatory roles for dALS2 in endosomal trafficking, synaptic development, and neuronal survival.     

Mitosis in Neurons: Roughex and APC/C Maintain Cell Cycle Exit to Prevent Cytokinetic and Axonal Defects in Drosophila Photoreceptor Neurons

The mechanisms of cell cycle exit by neurons remain poorly understood. Through genetic and developmental analysis of Drosophila eye development, we found that the cyclin-dependent kinase-inhibitor Roughex maintains G1 cell cycle exit during differentiation of the R8 class of photoreceptor neurons. The roughex mutant neurons re-enter the mitotic cell cycle and progress without executing cytokinesis, unlike non-neuronal cells in the roughex mutant that perform complete cell divisions. After mitosis, the binucleated R8 neurons usually transport one daughter nucleus away from the cell body into the developing axon towards the brain in a kinesin-dependent manner resembling anterograde axonal trafficking. Similar cell cycle and photoreceptor neuron defects occurred in mutants for components of the Anaphase Promoting Complex/Cyclosome. These findings indicate a neuron-specific defect in cytokinesis and demonstrate a critical role for mitotic cyclin downregulation both to maintain cell cycle exit during neuronal differentiation and to prevent axonal defects following failed cytokinesis.     

Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain

Background

Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.

Methodology/Principal Findings

In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.

Conclusions/Significance

Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons.     

Metabolome Analysis of Drosophila melanogaster during Embryogenesis

The Drosophila melanogaster embryo has been widely utilized as a model for genetics and developmental biology due to its small size, short generation time, and large brood size. Information on embryonic metabolism during developmental progression is important for further understanding the mechanisms of Drosophila embryogenesis. Therefore, the aim of this study is to assess the changes in embryos’ metabolome that occur at different stages of the Drosophila embryonic development. Time course samples of Drosophila embryos were subjected to GC/MS-based metabolome analysis for profiling of low molecular weight hydrophilic metabolites, including sugars, amino acids, and organic acids. The results showed that the metabolic profiles of Drosophila embryo varied during the course of development and there was a strong correlation between the metabolome and different embryonic stages. Using the metabolome information, we were able to establish a prediction model for developmental stages of embryos starting from their high-resolution quantitative metabolite composition. Among the important metabolites revealed from our model, we suggest that different amino acids appear to play distinct roles in different developmental stages and an appropriate balance in trehalose-glucose ratio is crucial to supply the carbohydrate source for the development of Drosophila embryo.     

Divergent and conserved roles of extradenticle in body segmentation and appendage formation, respectively, in the cricket Gryllus bimaculatus

The cricket Gryllus bimaculatus is a typical hemimetabolous intermediate germ insect, in which the processes of segmentation and appendage formation differ from those in Drosophila, a holometabolous long germ insect. In order to compare their developmental mechanisms, we have focused on Gryllus orthologs of the Drosophila developmental regulatory genes and studied their functions. Here, we report a functional analysis of the Gryllus ortholog of extradenticle (Gb′exd) using embryonic and parental RNA interference (RNAi) techniques. We found the following: (1) RNAi suppression of Gb′exd results in the deletion or fusion of body segments. Especially the head was often very severely affected. This gap-like phenotype may be related to reduced expression of the gap genes hunchback and Krüppel in early RNAi germbands. (2) In the appendages, several segments (podomeres) were fused. (3) Head appendages including the antenna were transformed to a leg-like structure consisting of at least one proximal podomere as well as several tarsomeres. The defects in appendages are reminiscent of the phenotype caused by large exd clones in Drosophila antennal discs. These findings led us to the conclusion that (1) Gb′exd is required for segment patterning in the gnathal to abdominal region, acting in a gap gene-like manner in the anterior region. (2) Gb′exd plays important roles in formation of the appendages and the determination of their identities, acting as a regulatory switch that chooses between the fates of head appendages versus the appendage ground state. Although functions of Gb′exd in appendage patterning appear fundamentally conserved between Gryllus and Drosophila, its role in body segmentation may differ from that of Drosophila exd.     

Prostaglandins regulate nuclear localization of Fascin and its function in nucleolar architecture

Fascin, a highly conserved actin-bundling protein, localizes and functions at new cellular sites in both Drosophila and multiple mammalian cell types. During Drosophila follicle development, in addition to being cytoplasmic, Fascin is in the nuclei of the germline-derived nurse cells during stages 10B–12 (S10B–12) and at the nuclear periphery during stage 13 (S13). This localization is specific to Fascin, as other actin-binding proteins, Villin and Profilin, do not exhibit the same subcellular distribution. In addition, localization of fascin1 to the nucleus and nuclear periphery is observed in multiple mammalian cell types. Thus the regulation and function of Fascin at these new cellular locations is likely to be highly conserved. In Drosophila, loss of prostaglandin signaling causes a global reduction in nuclear Fascin and a failure to relocalize to the nuclear periphery. Alterations in nuclear Fascin levels result in defects in nucleolar morphology in both Drosophila follicles and cultured mammalian cells, suggesting that nuclear Fascin plays an important role in nucleolar architecture. Given the numerous roles of Fascin in development and disease, including cancer, our novel finding that Fascin has functions within the nucleus sheds new light on the potential roles of Fascin in these contexts.     

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of <8 hours (at 25°C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.Open in a separate windowClick here to view.^{(51M, flv)}     

The histone lysine methyltransferase Ezh2 is required for maintenance of the intestine integrity and for caudal fin regeneration in zebrafish

The histone lysine methyltransferase EZH2, as part of the Polycomb Repressive Complex 2 (PRC2), mediates H3K27me3 methylation which is involved in gene expression program repression. Through its action, EZH2 controls cell-fate decisions during the development and the differentiation processes. Here, we report the generation and the characterization of an ezh2-deficient zebrafish line. In contrast to its essential role in mouse early development, loss of ezh2 function does not affect zebrafish gastrulation. Ezh2 zebrafish mutants present a normal body plan but die at around 12 dpf with defects in the intestine wall, due to enhanced cell death. Thus, ezh2-deficient zebrafish can initiate differentiation toward the different developmental lineages but fail to maintain the intestinal homeostasis. Expression studies revealed that ezh2 mRNAs are maternally deposited. Then, ezh2 is ubiquitously expressed in the anterior part of the embryos at 24 hpf, but its expression becomes restricted to specific regions at later developmental stages. Pharmacological inhibition of Ezh2 showed that maternal Ezh2 products contribute to early development but are dispensable to body plan formation. In addition, ezh2-deficient mutants fail to properly regenerate their spinal cord after caudal fin transection suggesting that Ezh2 and H3K27me3 methylation might also be involved in the process of regeneration in zebrafish.     

Heat Shock during the Development of Central Structures of the Drosophila Brain: Memory Formation in the l(1)ts403 Mutant of Drosophila melanogaster

The structures and functions of many genes are homologous in Drosophilaand humans. Therefore, studying pathological processes in Drosophila, in particular neurogenerative processes accompanied by progressive memory loss, helps to understand the ethiology of corresponding human disorders and to develop therapeutic strategies. It is believed that the development of neurogenerative diseases might result from alterations in the functioning of the heat shock/chaperone machinery. In view of this, we used Drosophila mutant l(1)ts403 with defective synthesis of heat shock proteins for studying learning and memory in a test of conditioned courtship suppression following a heat shock given at different developmental stages. High learning indices were registered immediately and 30 min after training both in the intact controls and in flies subjected to different developmental heat shocks. This indicated normal learning and memory acquisition in the mutant. At the same time, memory retention (3 h after training) suffered to different extent depending on the developmental stage. The remote effects of heat shock given during the formation of the mushroom bodies indicated the important role of this brain structure in the memory formation. The observed memory defects may result from alterations both in mRNA transport and in the functions of molecular chaperones in the l(1)ts403 mutant.     

Conserved genetic programs in insect and mammalian brain development

In recent years it has become evident that the developmental regulatory genes involved in patterning the embryonic body plan are conserved throughout the animal kingdom. Striking examples are the orthodenticle (otd/Otx) gene family and the Hox gene family, both of which act in the specification of anteroposterior polarity along the embryonic body axis. Studies carried out in Drosophila and mouse now demonstrate that these genes are also involved in the formation of the insect and mammalian brain; the otd/Otx genes are involved in rostral brain development and the Hox genes are involved in caudal brain development. These studies also show that the genes of the otd/Otx family can functionally replace each other in cross-phylum rescue experiments and indicate that the genetic mechanisms underlying pattern formation in insect and mammalian brain development are evolutionarily conserved. BioEssays 21:677–684, 1999. © 1999 John Wiley & Sons, Inc.     

Apical constriction: A cell shape change that can drive morphogenesis

Biologists have long recognized that dramatic bending of a cell sheet may be driven by even modest shrinking of the apical sides of cells. Cell shape changes and tissue movements like these are at the core of many of the morphogenetic movements that shape animal form during development, driving processes such as gastrulation, tube formation, and neurulation. The mechanisms of such cell shape changes must integrate developmental patterning information in order to spatially and temporally control force production—issues that touch on fundamental aspects of both cell and developmental biology and on birth defects research. How does developmental patterning regulate force-producing mechanisms, and what roles do such mechanisms play in development? Work on apical constriction from multiple systems including Drosophila, Caenorhabditis elegans, sea urchin, Xenopus, chick, and mouse has begun to illuminate these issues. Here, we review this effort to explore the diversity of mechanisms of apical constriction, the diversity of roles that apical constriction plays in development, and the common themes that emerge from comparing systems.     

Requirement for Dlgh-1 in Planar Cell Polarity and Skeletogenesis during Vertebrate Development

The development of specialized organs is tightly linked to the regulation of cell growth, orientation, migration and adhesion during embryogenesis. In addition, the directed movements of cells and their orientation within the plane of a tissue, termed planar cell polarity (PCP), appear to be crucial for the proper formation of the body plan. In Drosophila embryogenesis, Discs large (dlg) plays a critical role in apical-basal cell polarity, cell adhesion and cell proliferation. Craniofacial defects in mice carrying an insertional mutation in Dlgh-1 suggest that Dlgh-1 is required for vertebrate development. To determine what roles Dlgh-1 plays in vertebrate development, we generated mice carrying a null mutation in Dlgh-1. We found that deletion of Dlgh-1 caused open eyelids, open neural tube, and misorientation of cochlear hair cell stereociliary bundles, indicative of defects in planar cell polarity (PCP). Deletion of Dlgh-1 also caused skeletal defects throughout the embryo. These findings identify novel roles for Dlgh-1 in vertebrates that differ from its well-characterized roles in invertebrates and suggest that the Dlgh-1 null mouse may be a useful animal model to study certain human congenital birth defects.