Helen Skaer

Secretion and movement of wingless protein in the epidermis of the Drosophila embryo

The segment polarity gene wingless encodes a cysteine rich protein which is essential for pattern formation in Drosophila. Using polyclonal antibodies against the product of the wingless gene, we demonstrate that this protein is secreted in the embryo and that it is taken up by neighbouring cells. The protein can be found two or three cell diameters away from the cells in which it is synthesized. We discuss the possible mechanisms which are responsible for this spatial distribution and its regulation during embryogenesis.

The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm

The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation, ‘nephron-like’ features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors. Here we show that the insect nephrocyte has remarkable anatomical, molecular and functional similarity to the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialized filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. We find that fly (Drosophila melanogaster) orthologues of the major constituents of the slit diaphragm, including nephrin, NEPH1 (also known as KIRREL), CD2AP, ZO-1 (TJP1) and podocin, are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, we find that the nephrocyte diaphragm is completely lost in flies lacking the orthologues of nephrin or NEPH1—a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in human congenital nephrotic syndrome of the Finnish type, NPHS1) or NEPH1. These changes markedly impair filtration function in the nephrocyte. The similarities we describe between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting that the two cell types are evolutionarily related, and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated diseases.

The Developmental, Molecular, and Transport Biology of Malpighian Tubules

Molecular biology is reaching new depths in our understanding of the development and physiology of Malpighian tubules. In Diptera, Malpighian tubules derive from ectodermal cells that evaginate from the primitive hindgut and subsequently undergo a sequence of orderly events that culminates in an active excretory organ by the time the larva takes its first meal. Thereafter, the tubules enlarge by cell growth. Just as modern experimental strategies have illuminated the development of tubules, genomic, transcriptomic, and proteomic studies have uncovered new tubule functions that serve immune defenses and the breakdown and renal clearance of toxic substances. Moreover, genes associated with specific diseases in humans are also found in flies, some of which, astonishingly, express similar pathophenotypes. However, classical experimental approaches continue to show their worth by distinguishing between -omic possibilities and physiological reality while providing further detail about the rapid regulation of the transport pathway through septate junctions and the reversible assembly of proton pumps.

Polymer cryoprotectants in the preservation of biological ultrastructure. I. Low temperature states of aqueous solutions of hydrophilic polymers.

The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoproteetants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspherical morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.The solid states formed by vitrified and frozen aqueous solutions of some hydrophilic polymers, able to act as biological cryoprotectants, have been studied by differential scanning calorimetry and freeze fracture electron microscopy. Glass transitions, devitrification, recrystallization and melting behaviour of aqueous solutions of polyvinylpyrrolidone, hydroxyethyl starch and dextran have been established. The vitrified polymer solutions exhibit a characteristic microspheral morphology which is not induced by the quench cooling process but is an inherent feature of the solutions themselves.

Polymeric cryoproteetants in the preservation of biological ultrastructure

A study has been made of the physiological effects of three non‐penetrating polymeric cryoprotective agents on sixteen different plant and animal cells and tissues. The cryoproteetants, when used at concentrations at which they are effective in preventing ice‐crystal formation, generally have a lower toxicity to cells and tissue than similar concentrations of glycerol. The relatively low toxicity of these substances suggests that they would be more suitable as cryoproteetants for morphological and analytical studies than the commonly used low molecular weight compounds.

Compartmentalisation of Rho regulators directs cell invagination during tissue morphogenesis

During development, small RhoGTPases control the precise cell shape changes and movements that underlie morphogenesis. Their activity must be tightly regulated in time and space, but little is known about how Rho regulators (RhoGEFs and RhoGAPs) perform this function in the embryo. Taking advantage of a new probe that allows the visualisation of small RhoGTPase activity in Drosophila, we present evidence that Rho1 is apically activated and essential for epithelial cell invagination, a common morphogenetic movement during embryogenesis. In the posterior spiracles of the fly embryo, this asymmetric activation is achieved by at least two mechanisms: the apical enrichment of Rho1; and the opposing distribution of Rho activators and inhibitors to distinct compartments of the cell membrane. At least two Rho1 activators, RhoGEF2 and RhoGEF64C are localised apically, whereas the Rho inhibitor RhoGAP Cv-c localises at the basolateral membrane. Furthermore, the mRNA of RhoGEF64C is also apically enriched, depending on signals present within its open reading frame, suggesting that apical transport of RhoGEF mRNA followed by local translation is a mechanism to spatially restrict Rho1 activity during epithelial cell invagination.

Dual origin of the renal tubules in Drosophila: mesodermal cells integrate and polarize to establish secretory function.

Organs are made up of cells from separate origins, whose development and differentiation must be integrated to produce a physiologically coherent structure. For example, during the development of the kidney, a series of interactions between the epithelial mesonephric duct and the surrounding metanephric mesenchyme leads to the formation of renal tubules. Cells of the metanephric mesenchyme first induce branching of the mesonephric duct to form the ureteric buds, and they then respond to signals derived from them. As a result, mesenchymal cells are recruited to the buds, where they undergo a mesenchymal-to-epithelial transition as they condense to form nephrons. In contrast, the simple renal tubules of invertebrates, such as insect Malpighian tubules (MpTs), have always been thought to arise from single tissue primordia, epithelial buds that grow by cell division and enlargement and from which a range of specialized subtypes differentiate. Here, we reveal unexpected parallels between the development of Drosophila MpTs and vertebrate nephrogenesis by showing that the MpTs also derive from two cell populations: ectodermal epithelial buds and the surrounding mesenchymal mesoderm. The mesenchymal cells are recruited to the growing tubules, where they undergo a mesenchymal-to-epithelial transition as they integrate and subsequently differentiate as a physiologically distinctive subset of tubule cells, the stellate cells. Strikingly, the normal incorporation of stellate cells and the later physiological activity of the mature tubules depend on the activity of hibris, an ortholog of mammalian NEPHRIN.

Hemocyte-Secreted Type IV Collagen Enhances BMP Signaling to Guide Renal Tubule Morphogenesis in Drosophila

Summary Details of the mechanisms that determine the shape and positioning of organs in the body cavity remain largely obscure. We show that stereotypic positioning of outgrowing Drosophila renal tubules depends on signaling in a subset of tubule cells and results from enhanced sensitivity to guidance signals by targeted matrix deposition. VEGF/PDGF ligands from the tubules attract hemocytes, which secrete components of the basement membrane to ensheath them. Collagen IV sensitizes tubule cells to localized BMP guidance cues. Signaling results in pathway activation in a subset of tubule cells that lead outgrowth through the body cavity. Failure of hemocyte migration, loss of collagen IV, or abrogation of BMP signaling results in tubule misrouting and defective organ shape and positioning. Such regulated interplay between cell-cell and cell-matrix interactions is likely to have wide relevance in organogenesis and congenital disease.

Renal Tubule Development in Drosophila: A Closer Look at the Cellular Level

The function of excretion in insects is performed by the Malpighian tubules, a functional equivalent of the vertebrate kidney. Malpighian tubules are long, thin tubes connected to the hindgut. Upon the determination of the Malpighian tubule major cell type early in embryogenesis, the tubular architecture is achieved by extensive cell division and cell rearrangements. During the tube elongation process, cells exchange their neighbors, allowing the short and fat Malpighian tubule primordia to grow and become a thin tube. Cell rearrangement and intercalation underlie the morphogenesis of other epithelial tissues in Drosophila melanogaster, such as the embryonic epidermis. Recent work has provided insights in the cellular and molecular basis of cell intercalation. These advances are reviewed and discussed with regard to what is known about Malpighian tubule morphogenesis. Mature Malpighian tubules are composed of two cell types, each having a specific function in excretion: The principal cells and the stellate cells. Drosophila and mammalian kidney development show striking similarities, as the recruitment of the stellate cells to the Malpighian tubules, like the cells of the metanephric mesenchyme, requires that cells undergo a mesenchymal-to-epithelial transition. The molecular similarities between these two cases is reviewed here.

Crumbs stabilises epithelial polarity during tissue remodelling.

The apicobasal polarity of epithelia depends on the integrated activity of apical and basolateral proteins, and is essential for tissue integrity and body homeostasis. Yet these tissues are frequently on the move as they are sculpted by active morphogenetic cell rearrangements. How does cell polarity survive these stresses? We analyse this question in the renal tubules of Drosophila, a tissue that undergoes dramatic morphogenetic change as it develops. Here we show that, whereas the Bazooka and Scribble protein groups are required for the establishment of tubule cell polarity, the key apical determinant, Crumbs, is required for cell polarity in the tubules only from the time when morphogenetic movements start. Strikingly, if these movements are stalled, polarity persists in the absence of Crumbs. Similar rescue of the ectodermal phenotype of the crumbs mutant when germ-band extension is reduced suggests that Crumbs has a specific, conserved function in stabilising cell polarity during tissue remodelling rather than in its initial stabilisation. We also identify a requirement for the exocyst component Exo84 during tissue morphogenesis, which suggests that Crumbs-dependent stability of epithelial polarity is correlated with a requirement for membrane recycling and targeted vesicle delivery.