Wnt-controlled sphingolipids modulate Anthrax Toxin Receptor palmitoylation to regulate oriented mitosis in zebrafish.

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis.

Anthrax toxin receptor 2a controls mitotic spindle positioning.

Oriented mitosis is essential during tissue morphogenesis. The Wnt/planar cell polarity (Wnt/PCP) pathway orients mitosis in a number of developmental systems, including dorsal epiblast cell divisions along the animal-vegetal (A-V) axis during zebrafish gastrulation. How Wnt signalling orients the mitotic plane is, however, unknown. Here we show that, in dorsal epiblast cells, anthrax toxin receptor 2a (Antxr2a) accumulates in a polarized cortical cap, which is aligned with the embryonic A-V axis and forecasts the division plane. Filamentous actin (F-actin) also forms an A-V polarized cap, which depends on Wnt/PCP and its effectors RhoA and Rock2. Antxr2a is recruited to the cap by interacting with actin. Antxr2a also interacts with RhoA and together they activate the diaphanous-related formin zDia2. Mechanistically, Antxr2a functions as a Wnt-dependent polarized determinant, which, through the action of RhoA and zDia2, exerts torque on the spindle to align it with the A-V axis.

The missing link: implementation of morphogenetic growth control on the cellular and molecular level.

In the wing imaginal disc of Drosophila melanogaster, the morphogen Dpp controls growth, probably in an instructive manner. Many models for growth control by Dpp have been proposed and have been extensively discussed elsewhere. In this review, we speculate on how instructive growth control could provide a link between Dpp signaling and cell growth and/or cell cycle progression and so implement morphogenetic growth control on the cellular and molecular levels.

Dynamics of Dpp signaling and proliferation control.

Morphogens, such as Decapentaplegic (Dpp) in the fly imaginal discs, form graded concentration profiles that control patterning and growth of developing organs. In the imaginal discs, proliferative growth is homogeneous in space, posing the conundrum of how morphogen concentration gradients could control position-independent growth. To understand the mechanism of proliferation control by the Dpp gradient, we quantified Dpp concentration and signaling levels during wing disc growth. Both Dpp concentration and signaling gradients scale with tissue size during development. On average, cells divide when Dpp signaling levels have increased by 50%. Our observations are consistent with a growth control mechanism based on temporal changes of cellular morphogen signaling levels. For a scaling gradient, this mechanism generates position-independent growth rates.

Criteria and outcome of limb salvage surgery.

When sufficient margins of resection surrounding the tumor can be achieved, limb salvage surgery, as opposed to amputation, has become the standard of care in treating patients with bone and soft tissue sarcoma of the extremities. Currently, 90-95% of patients with primary malignant bone and soft-tissue tumors involving the extremities can be treated safely with wide resection and limb salvage surgery with a low risk of recurrence and the same disease-free survival rate as amputative surgery. However, discussions persist regarding the indications and criteria, and whether limb salvage provides superior functional results and quality of life for cancer patients. In this study we aimed to review and update the current criteria, indications and contraindications of limb salvage surgery and discuss its role in the quality of life of cancer patients.

Quantification of growth asymmetries in developing epithelia.

Many developmental processes of multicellular organisms involve the patterning and growth of two-dimensional tissues, so called epithelia. We have quantified the growth of the wing imaginal disk, which is the precursor of the adult wing, of the fruit fly Drosophila melanogaster. We find that growth follows a simple rule with exponentially decreasing area growth rate. Anisotropies of growth can be precisely determined by comparing experimental results to a continuum theory. Growth anisotropies are to good approximation constant in space and time. They are weak in wild-type wing disks but threefold increased in GFP-Dpp disks in which the morphogen Dpp is overexpressed. Our findings indicate that morphogens such as Dpp control tissue shape via oriented cell divisions that generate anisotropic growth.

Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division.

Endocytosis has a crucial role during Notch signalling after the asymmetric division of fly sensory organ precursors (SOPs): directional signalling is mediated by differential endocytosis of the ligand Delta and the Notch effector Sanpodo in one of the SOP daughters, pIIb. Here we show a new mechanism of directional signalling on the basis of the trafficking of Delta and Notch molecules already internalized in the SOP and subsequently targeted to the other daughter cell, pIIa. Internalized Delta and Notch traffic to an endosome marked by the protein Sara. During SOP mitosis, Sara endosomes containing Notch and Delta move to the central spindle and then to pIIa. Subsequently, in pIIa (but not in pIIb) Notch appears cleaved in Sara endosomes in a gamma-secretase- and Delta internalization-dependent manner, indicating that the release of the intracellular Notch tail to activate Notch target genes has occurred. We thus uncover a new mechanism to bias signalling even before asymmetric endocytosis of Sanpodo and Delta takes place in the daughter cells: already during SOP mitosis, asymmetric targeting of Delta and Notch-containing Sara endosomes will increase Notch signalling in pIIa and decrease it in pIIb.

Morphogen transport in epithelia.

We present a general theoretical framework to discuss mechanisms of morphogen transport and gradient formation in a cell layer. Trafficking events on the cellular scale lead to transport on larger scales. We discuss in particular the case of transcytosis where morphogens undergo repeated rounds of internalization into cells and recycling. Based on a description on the cellular scale, we derive effective nonlinear transport equations in one and two dimensions which are valid on larger scales. We derive analytic expressions for the concentration dependence of the effective diffusion coefficient and the effective degradation rate. We discuss the effects of a directional bias on morphogen transport and those of the coupling of the morphogen and receptor kinetics. Furthermore, we discuss general properties of cellular transport processes such as the robustness of gradients and relate our results to recent experiments on the morphogen Decapentaplegic (Dpp) that acts in the wing disk of the fruit fly Drosophila.

Robust formation of morphogen gradients.

We discuss the formation of graded morphogen profiles in a cell layer by nonlinear transport phenomena, important for patterning developing organisms. We focus on a process termed transcytosis, where morphogen transport results from the binding of ligands to receptors on the cell surface, incorporation into the cell, and subsequent externalization. Starting from a microscopic model, we derive effective transport equations. We show that, in contrast to morphogen transport by extracellular diffusion, transcytosis leads to robust ligand profiles which are insensitive to the rate of ligand production.

Gradient formation of the TGF-beta homolog Dpp.

Secreted morphogens such as the Drosophila TGF-beta homolog Decapentaplegic (Dpp) are thought to spread through target tissues and form long-range concentration gradients providing positional information. Using a GFP-Dpp fusion, we monitored a TGF-beta family member trafficking in situ throughout the target tissue and forming a long-range concentration gradient. Evidence is presented that long-range Dpp movement involves Dpp receptor and Dynamin functions. We also show that the rates of endocytic trafficking and degradation determine Dpp signaling range. We propose a model where the gradient is formed via intracellular trafficking initiated by receptor-mediated endocytosis of the ligand in receiving cells with the gradient slope controlled by endocytic sorting of Dpp toward recycling versus degradation.

Tip cell-derived RTK signaling initiates cell movements in the Drosophila stomatogastric nervous system anlage.

The stomatogastric nervous system (SNS) of Drosophila is a simply organized neural circuitry that innervates the anterior enteric system. Unlike the central and the peripheral nervous systems, the SNS derives from a compact epithelial anlage in which three invagination centers, each giving rise to an invagination fold headed by a tip cell, are generated. Tip cell selection involves lateral inhibition, a process in which Wingless (Wg) activity adjusts the range of Notch signaling. Here we show that RTK signaling mediated by the Drosophila homolog of the epidermal growth factor receptor, DER, plays a key role in two consecutive steps during early SNS development. Like Wg, DER signaling participates in adjusting the range of Notch-dependent lateral inhibition during tip cell selection. Subsequently, tip cells secrete the DER ligand Spitz and trigger local RTK signaling, which initiates morphogenetic movements resulting in the tip cell-directed invaginations within the SNS anlage.

The range of spalt-activating Dpp signalling is reduced in endocytosis-defective Drosophila wing discs.

Pattern formation along the anterior-posterior (A/P) axis of the developing Drosophila wing depends on Decapentaplegic (Dpp), a member of the conserved transforming growth factor beta (TGFbeta) family of secreted proteins. Dpp is expressed in a stripe along the A/P compartment boundary of the wing imaginal disc and forms a long-range concentration gradient with morphogen-like properties which generates distinct cell fates along the A/P axis. We have monitored Dpp expression and Dpp signalling in endocytosis-mutant wing imaginal discs which develop severe pattern defects specifically along the A/P wing axis. The results show that the size of the Dpp expression domain is expanded in endocytosis-mutant wing discs. However, this expansion did not result in a concomitant expansion of the functional range of Dpp activity but rather its reduction as indicated by the reduced expression domain of the Dpp target gene spalt. The data suggest that clathrin-mediated endocytosis, a cellular process necessary for membrane recycling and vesicular trafficking, participates in Dpp action during wing development. Genetic interaction studies suggest a link between the Dpp receptors and clathrin. Impaired endocytosis does not interfere with the reception of the Dpp signal or the intracellular processing of the mediation of the signal in the responder cells, but rather affects the secretion and/or the distribution of Dpp in the developing wing cells.

Genetic analysis of stomatogastric nervous system development in Drosophila using enhancer trap lines.

The stomatogastric nervous system (SNS) of Drosophila melanogaster is a small, simply organized neural circuitry which innervates the anterior enteric system. It is responsible for regulating the passage of food through the pharynx and esophagus and into the midgut. Here we show that the development of the SNS is amenable to genetic dissection. We screened lines from a P-element mutagenesis, selecting those with lacZ reporter gene expression and/or a phenotype in the SNS, associated glia, and garland cells. We report a collection of expression patterns and mutant phenotypes among lines found to have a mutation in genes required for the establishment of the larval SNS. Our results indicate that SNS development depends on pattern organizer genes including components of the Ras/Raf pathway.

Role of Drosophila alpha-adaptin in presynaptic vesicle recycling.

Rapid flow of information in the nervous system involves presynaptic vesicle recycling by clathrin-mediated endocytosis, an event triggered by the alpha-adaptin-containing AP2 complex. We identified a Drosophila alpha-adaptin expressed in the garland cells, imaginal discs, and the CNS. Here we show its role in presynaptic vesicle recycling. In presynaptic terminals, alpha-adaptin defines a network-like membrane structure to which the GTPase dynamin is recruited. alpha-adaptin is necessary for the formation of clathrin-coated pits and participates in the dynamin-dependent release of coated vesicles from the membrane surface. Our results suggest an alpha-adaptin-dependent control of the vesicle cycle that maintains the balance between the amount of vesicle- and surface-associated membranes.

Invagination centers within the Drosophila stomatogastric nervous system anlage are positioned by Notch-mediated signaling which is spatially controlled through wingless.

The gut-innervating stomatogastric nervous system of Drosophila, unlike the central and the peripheral nervous system, derives from a compact, single layered epithelial anlage. Here we report how this anlage is initially defined during embryogenesis by the expression of proneural genes of the achaete-scute complex in response to the maternal terminal pattern forming system. Within the stomatogastric nervous system anlage, the wingless-dependent intercellular communication system adjusts the cellular range of Notch-dependent lateral inhibition to single-out three achaete-expressing cells. Those cells define distinct invagination centers which orchestrate the behavior of neighboring cells to form epithelial infoldings, each headed by an achaete-expressing tip cell. Our results suggest that the wingless pathway acts not as an instructive signal, but as a permissive factor which coordinates the spatial activity of morphoregulatory signals within the stomatogastric nervous system anlage.

Redundant functions of the genes knirps and knirps-related for the establishment of anterior Drosophila head structures.

Developmental gene functions of Drosophila are typically characterized by a recognizable mutant phenotype. When molecular probes of such genes were used to isolate homologues, distinct spatially and temporally restricted expression patterns were observed in vertebrates as well. However, corresponding "gene knock-outs" often revealed subtle or no scorable phenotypes, a phenomenon attributed to redundant gene functions. We found that the evolutionarily related genes knirps (kni) and knirps-related (knrl) contribute to a similar phenomenon in Drosophila. The two closely situated genes show identical expression patterns in the developing embryo, including the posterior and anterior expression domains in the blastoderm. Here we show that the two biochemically equivalent gene products are both functional in the head anlage and that the lack of one gene activity can be overcome by the activity of the other. Whereas kni is also required for abdominal segmentation, knrl is nonfunctional in its posterior expression domain. Thus, the kni/knrl pair of genes provides a region-specific buffering system, rather than a case of global functional redundancy.

Number, identity, and sequence of the Drosophila head segments as revealed by neural elements and their deletion patterns in mutants.

The development of the insect head tagma involves massive rearrangements and secondary fusions of segment anlagen during embryogenesis. Due to the lack of reliable morphological markers, the number, identity, and sequence of the head segments, particularly in the pregnathal region, are still a matter of ongoing debates. We examined the complex array of internal structures of the embryonic Drosophila melanogaster head such as the sensory structures and nerves of the peripheral and stomatogastric nervous systems, and we used embryonic head mutations causing a lack of overlapping segment anlagen to unravel the segmental identity and the sequence of the neural elements. Our results provide evidence for seven distinct segments in the Drosophila head, each characterized by a specific set of sensory neurons, consistent with the proposal that insects, myriapods, and crustaceans share a monophyletic evolutionary tree from a common annelid-like ancestor.

Identical transacting factor requirement for knirps and knirps-related Gene expression in the anterior but not in the posterior region of the Drosophila embryo.

The Drosophila genes knirps (kni) and knirps-related (knrl) are located within the 77E1,2 region on the left arm of the third chromosome. They encode nuclear hormone-like transcription factors containing almost identical Cys2/Cys2 DNA-binding zinc finger motifs which bind to the same target sequence. kni is a member of the gap class of segmentation genes, and its activity is required for the normal establishment of the abdomen. The function of knrl is still unknown; however, a possible gap gene function in the abdominal region of the embryo can be excluded. Both genes are initially expressed in three identical regions of the blastoderm embryo: in an anterior cap domain, in an anterior stripe and in a posterior broad band linked to the kni gap gene function. The transacting factor requirement for the expression of kni and knrl is identical for the two anterior domains but different, although similar, for the posterior domain of expression in the blastoderm. Both the anteroposterior morphogen bicoid and the dorsoventral morphogen dorsal are necessary but not sufficient for the activation of the two genes in the anterior cap domain, suggesting they act together to bring about its normal spatial limits.

Cell proliferation patterns in the wing imaginal disc of Drosophila.

Morphogenetic processes, based on the temporal and spatial control of cell proliferation, are involved in determining the size and shape of an organism. We have used clonal analysis, employing X-ray-induced mitotic recombination, to study cell proliferation and differentiation processes in the developing wing imaginal disc of Drosophila. Our results show a non-uniform distribution of mitotic activities during different stages of wing development. This may reflect waves of cell proliferation which derive from distinct centers of cell proliferation within the growing wing imaginal disc. These proliferation centers are located within the major wing compartments (i.e. the anterior, posterior, dorsal and ventral compartments) and they are restricted to the areas which give rise to the intervein regions of the adult wing. The mitotic recombination analysis, combined with the study of Minute and gynandromorph mosaics, show that the presumptive vein regions of the wing represent distinct boundaries which delimit the proliferation centers to the intervein regions. We present a generative model of wing morphogenesis that is consistent with our results.

spalt encodes an evolutionarily conserved zinc finger protein of novel structure which provides homeotic gene function in the head and tail region of the Drosophila embryo.

The region specific homeotic gene spalt (sal) of Drosophila melanogaster promotes the specification of terminal pattern elements as opposed to segments in the trunk. Our results show that the previously reported sal transcription unit was misidentified. Based on P-element mediated germ line transformation and DNA sequence analysis of sal mutant alleles, we identified the transcription unit that carries sal function. sal is located close to the misidentified transcription unit, and it is expressed in similar temporal and spatial patterns during embryogenesis. The sal gene encodes a zinc finger protein of novel structure composed of three widely spaced 'double zinc finger' motifs of internally conserved sequences and a single zinc finger motif of different sequence. Antibodies produced against the sal protein show that sal is first expressed at the blastoderm stage and later in restricted areas of the embryonic nervous system as well as in the developing trachea. The antibodies detect sal homologous proteins in corresponding spatial and temporal patterns in the embryos of related insect species. Sequence analysis of the sal gene of Drosophila virilis, a species which is phylogenetically separated by approximately 60 million years, suggests that the sal function is conserved during evolution, consistent with its proposed role in head formation during arthropod evolution.