Protein crosslinking by transglutaminase controls cuticle morphogenesis in Drosophila

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins--two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin-as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.     

The extracellular matrix in development and morphogenesis: A dynamic view

The extracellular matrix (ECM) is synthesized and secreted by embryonic cells beginning at the earliest stages of development. Our understanding of ECM composition, structure and function has grown considerably in the last several decades and this knowledge has revealed that the extracellular microenvironment is critically important for cell growth, survival, differentiation and morphogenesis. ECM and the cellular receptors that interact with it mediate both physical linkages with the cytoskeleton and the bidirectional flow of information between the extracellular and intracellular compartments. This review considers the range of cell and tissue functions attributed to ECM molecules and summarizes recent findings specific to key developmental processes. The importance of ECM as a dynamic repository for growth factors is highlighted along with more recent studies implicating the 3-dimensional organization and physical properties of the ECM as it relates to cell signaling and the regulation of morphogenetic cell behaviors. Embryonic cell and tissue generated forces and mechanical signals arising from ECM adhesion represent emerging areas of interest in this field.     

The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis

The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.     

The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis

Long-chain acyl-CoA synthetase (LACS) activities are encoded by a family of at least nine genes in Arabidopsis (Arabidopsis thaliana). These enzymes have roles in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. Here, we show that the LACS2 gene (At1g49430) is expressed in young, rapidly expanding tissues, and in leaves expression is limited to cells of the adaxial and abaxial epidermal layers, suggesting that the LACS2 enzyme may act in the synthesis of cutin or cuticular waxes. A lacs2 null mutant was isolated by reverse genetics. Leaves of mutant plants supported pollen germination and released chlorophyll faster than wild-type leaves when immersed in 80% ethanol, indicating a defect in the cuticular barrier. The composition of surface waxes extracted from lacs2 leaves was similar to the wild type, and the total wax load was higher than the wild type (111.4 microg/dm(2) versus 76.4 microg/dm(2), respectively). However, the thickness of the cutin layer on the abaxial surface of lacs2 leaves was only 22.3 +/- 1.7 nm compared with 33.0 +/- 2.0 nm for the wild type. In vitro assays showed that 16-hydroxypalmitate was an excellent substrate for recombinant LACS2 enzyme. We conclude that the LACS2 isozyme catalyzes the synthesis of omega-hydroxy fatty acyl-CoA intermediates in the pathway to cutin synthesis. The lacs2 phenotype, like the phenotypes of some other cutin mutants, is very pleiotropic, causing reduced leaf size and plant growth, reduced seed production, and lower rates of seedling germination and establishment. The LACS2 gene and the corresponding lacs2 mutant will help in future studies of the cutin synthesis pathway and in understanding the consequences of reduced cutin production on many aspects of plant biology.     

The gene ENHANCER OF PINOID controls cotyledon development in the Arabidopsis embryo

During Arabidopsis embryo development, cotyledon primordia are generated at transition stage from precursor cells that are not derived from the embryonic shoot apical meristem (SAM). To date, it is not known which genes specifically instruct these precursor cells to elaborate cotyledons, nor is the role of auxin in cotyledon development clear. In laterne mutants, the cotyledons are precisely deleted, yet the hypocotyl and root are unaffected. The laterne phenotype is caused by a combination of two mutations: one in the PINOID (PID) gene and another mutation in a novel locus designated ENHANCER OF PINOID (ENP). The expression domains of shoot apex organising genes such as SHOOT MERISTEMLESS (STM) extend along the entire apical region of laterne embryos. However, analysis of pid enp stm triple mutants shows that ectopic activity of STM does not appear to cause cotyledon obliteration. This is exclusively caused by enp in concert with pid. In pinoid embryos, reversal of polarity of the PIN1 auxin transport facilitator in the apex is only occasional, explaining irregular auxin maxima in the cotyledon tips. By contrast, polarity of PIN1:GFP is completely reversed to basal position in the epidermal layer of the laterne embryo. Consequently auxin, which is believed to be essential for organ formation, fails to accumulate in the apex. This strongly suggests that ENP specifically regulates cotyledon development through control of PIN1 polarity in concert with PID.     

Cell morphogenesis in Arabidopsis

Cell morphogenesis encompasses all processes required to establish a three-dimensional cell shape. Cells acquire the architecture specific to their developmental context by using the spatial information provided by internal or external cues. As a response to these signals, cells become reorganized and establish functionally distinct subcellular domains that ultimately lead to morphological changes. In its simplest form, cell morphogenesis results in the establishment of asymmetry along one axis, a cell polarity. Although cell polarity has been studied intensively in budding yeast and epithelial cells, little is known about more complex modes of cell morphogenesis involving multiple axes. In this review we compare the regulation of cell morphogenesis of different genetically well-characterized cell types in Arabidopsis thaliana. BioEssays 20:20–29, 1998. © 1998 John Wiley & Sons, Inc.     

Trichome morphogenesis in Arabidopsis

Trichomes (plant hairs) in Arabidopsis thaliana are large non-secreting epidermal cells with a characteristic three-dimensional architecture. Because trichomes are easily accessible to a combination of genetic, cell biological and molecular methods they have become an ideal model system to study various aspects of plant cell morphogenesis. In this review we will summarize recent progress in the understanding of trichome morphogenesis.     

The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement

In the present study, we investigated a new member of the ABC transporter superfamily of Arabidopsis thaliana, AtMRP5. AtMRP5 encodes a 167 kDa protein and exhibits low glutathione conjugate and glucuronide conjugate transport activity. Promotor- beta-glucuronidase fusion constructs showed that AtMRP5 is expressed mainly in the vascular bundle and in the epidermis, especially guard cells. Using reverse genetics, we identified a plant with a T-DNA insertion in AtMRP5 (mrp5-1). mrp5-1 exhibited decreased root growth and increased lateral root formation. Auxin levels in the roots of mrp5-1 plants were increased. This observation may indicate that AtMRP5 works as an auxin conjugate transporter or that mutant plants are affected in ion uptake, which may lead to changes in auxin concentrations. Experiments on epidermal strips showed that in contrast to wild type, the sulfonylurea glibenclamide had no effect on stomatal opening in mrp5-1 plants. This result strongly suggests that AtMRP5 may also function as an ion channel regulator.     

The coiled-coil protein shrub controls neuronal morphogenesis in Drosophila

The diversity of neuronal cells, especially in the size and shape of their dendritic and axonal arborizations, is a striking feature of the mature nervous system. Dendritic branching is a complex process, and the underlying signaling mechanisms remain to be further defined at the mechanistic level. Here we report the identification of shrub mutations that increased dendritic branching. Single-cell clones of shrub mutant dendritic arborization (DA) sensory neurons in Drosophila larvae showed ectopic dendritic and axonal branching, indicating a cell-autonomous function for shrub in neuronal morphogenesis. shrub encodes an evolutionarily conserved coiled-coil protein homologous to the yeast protein Snf7, a key component in the ESCRT-III (endosomal sorting complex required for transport) complex that is involved in the formation of endosomal compartments known as multivesicular bodies (MVBs). We found that mouse orthologs could substitute for Shrub in mutant Drosophila embryos and that loss of Shrub function caused abnormal distribution of several early or late endosomal markers in DA sensory neurons. Our findings demonstrate that the novel coiled-coil protein Shrub functions in the endosomal pathway and plays an essential role in neuronal morphogenesis.     

The role of extracellular matrix in vascular branching morphogenesis

Angiogenesis requires the development of a hierarchically branched network of vessels, which undergoes radial expansion and anastomosis to form a close circuit. Branching is achieved by coordinated behavior of endothelial cells that organize into leading “tip” cells and trailing “stalk” cells. Such organization is under control of the Dll4-Notch signaling pathway, which sets a hierarchy in receptiveness of cells to VEGF-A. Recent studies have shed light on a control of the Notch pathway by basement membrane proteins and integrin signaling, disclosing that extracellular matrix exerts active control on vascular branching morphogenesis. We will survey in the present review how extracellular matrix is a multifaceted substrate, which behind a classical structural role hides a powerful conductor function to shape the branching pattern of vessels.     

Mutations in the FASS gene uncouple pattern formation and morphogenesis in Arabidopsis development.

The pattern of cell division is very regular in Arabidopsis embryogenesis, enabling seedling structures to be traced back to groups of cells in the early embryo. Recessive mutations in the FASS gene alter the pattern of cell division from the zygote, without interfering with embryonic pattern formation: although no primordia of seedling structures can be recognised by morphological criteria at the early-heart stage, all elements of the body pattern are differentiated in the seedling. fass seedlings are strongly compressed in the apical-basal axis and enlarged circumferentially, notably in the hypocotyl. Depending on the width of the hypocotyl, fass seedlings may have up to three supernumerary cotyledons. fass mutants can develop into tiny adult plants with all parts, including floral organs, strongly compressed in their longitudinal axis. At the cellular level, fass mutations affect cell elongation and orientation of cell walls but do not interfere with cell polarity as evidenced by the unequal division of the zygote. The results suggest that the FASS gene is required for morphogenesis, i.e., oriented cell divisions and position-dependent cell shape changes generating body shape, but not for cell polarity which seems essential for pattern formation.     

The development of the cuticle in Phormium tenax

Summary As seen in the scanning electron microscope the surface wax of leaves of Phormium tenax L. consists of vertical, plate-like crystals. These increase in size and number and undergo a change in form during development. The abaxial surface has a dense covering of wax crystals, but none are present on the ridges over vascular tissues. Numerous papillae are found between these ridges in later stages of development. On the adaxial surface both wax crystals and papillae are present only around infrequent stomata.When viewed in section normal to the leaf surface the cuticle is first apparent as a thin, lamellate layer. Another layer containing a reticulum of electrondense material increases in thickness beneath the lamellae during development. This layer eventually becomes the most extensive component of the cuticle. Both the adaxial and abaxial cuticles show a similar pattern of development.     

The role of extracellular matrix in vascular branching morphogenesis

Angiogenesis requires the development of a hierarchically branched network of vessels, which undergoes radial expansion and anastomosis to form a close circuit. Branching is achieved by coordinated behavior of endothelial cells that organize into leading “tip” cells and trailing “stalk” cells. Such organization is under control of the Dll4-Notch signaling pathway, which sets a hierarchy in receptiveness of cells to VEGF-A. Recent studies have shed light on a control of the Notch pathway by basement membrane proteins and integrin signaling, disclosing that extracellular matrix exerts active control on vascular branching morphogenesis. We will survey in the present review how extracellular matrix is a multifaceted substrate, which behind a classical structural role hides a powerful conductor function to shape the branching pattern of vessels.     

Dermal morphogenesis controls lateral line patterning during postembryonic development of teleost fish

The lateral line system displays highly divergent patterns in adult teleost fish. The mechanisms underlying this variability are poorly understood. Here, we demonstrate that the lateral line mechanoreceptor, the neuromast, gives rise to a series of accessory neuromasts by a serial budding process during postembryonic development in zebrafish. We also show that accessory neuromast formation is highly correlated to the development of underlying dermal structures such as bones and scales. Abnormalities in opercular bone morphogenesis, in endothelin 1-knockdown embryos, are accompanied by stereotypic errors in neuromast budding and positioning, further demonstrating the tight correlation between the patterning of neuromasts and of the underlying dermal bones. In medaka, where scales form between peridermis and opercular bones, the lateral line displays a scale-specific pattern which is never observed in zebrafish. These results strongly suggest a control of postembryonic neuromast patterns by underlying dermal structures. This dermal control may explain some aspects of the evolution of lateral line patterns.