Notch signaling generates the "cut here line" on the cuticle of the puparium in Drosophila melanogaster.

During a molt or eclosion, insects shed their cuticle, an extracellular matrix made by underlying epidermal cells, by cleavage along a defined line. This means that the "cut here line" is pre-formed on the cuticle, and its formation is indispensable for insect life. Here, we show that the proper formation of the operculum ridge (OR), which is the "cut here line" on the puparium (pupal case) of Drosophila melanogaster, involves Notch signaling activation in the epidermal cells just beneath the future OR region (OR-forming cells). The inhibition of Notch signaling causes defects in eclosion due to failure in OR cleavage, the chitin organization and several cuticular proteins localization, glucose dehydrogenase (Gld) activity, and OR-forming cell shape. Our findings provide the first insight into the molecular basis of the structure and formation of the "cut here line" on the cuticle.

A corset function of exoskeletal ECM promotes body elongation in Drosophila.

Body elongation is a general feature of development. Postembryonically, the body needs to be framed and protected by extracellular materials, such as the skeleton, the skin and the shell, which have greater strength than cells. Thus, body elongation after embryogenesis must be reconciled with those rigid extracellular materials. Here we show that the exoskeleton (cuticle) coating the Drosophila larval body has a mechanical property to expand less efficiently along the body circumference than along the anteroposterior axis. This "corset" property of the cuticle directs a change in body shape during body growth from a relatively round shape to an elongated one. Furthermore, the corset property depends on the functions of Cuticular protein 11 A and Tubby, protein components of a sub-surface layer of the larval cuticle. Thus, constructing a stretchable cuticle and supplying it with components that confer circumferential stiffness is the fly's strategy for executing postembryonic body elongation.

Cuticle itself as a central and dynamic player in shaping cuticle.

The wide variety of external morphologies has underlain the evolutionary success of insects. The insect exoskeleton, or cuticle, which covers the entire body and constitutes the external morphology, is extracellular matrix produced by the epidermis. How is cuticle shaped during development? Past studies have mainly focused on patterning, differentiation and morphogenesis of the epidermis. Recently, however, it is becoming clear that cuticle itself plays important and active roles in regulation of cuticle shape. Studies in the past several years show that pre-existing cuticle can influence shaping of new cuticle, and cuticle can sculpt its own shape through its material property. In this review, I summarize recent advances and discuss future prospects.

Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster.

Body shapes are much more variable than body plans. One way to alter body shapes independently of body plans would be to mechanically deform bodies. To what extent body shapes are regulated physically, or molecules involved in physical control of morphogenesis, remain elusive. During fly metamorphosis, the cuticle (exoskeleton) covering the larval body contracts longitudinally and expands laterally to become the ellipsoidal pupal case (puparium). Here we show that Drosophila melanogaster Obstructor-E (Obst-E) is a protein constituent of the larval cuticle that confers the oriented contractility/expandability. In the absence of obst-E function, the larval cuticle fails to undergo metamorphic shape change and finally becomes a twiggy puparium. We present results indicating that Obst-E regulates the arrangement of chitin, a long-chain polysaccharide and a central component of the insect cuticle, and directs the formation of supracellular ridges on the larval cuticle. We further show that Obst-E is locally required for the oriented shape change of the cuticle during metamorphosis, which is associated with changes in the morphology of those ridges. Thus, Obst-E dramatically affects the body shape in a direct, physical manner by controlling the mechanical property of the exoskeleton.

Progressive tarsal patterning in the Drosophila by temporally dynamic regulation of transcription factor genes.

The morphology of insect appendages, such as the number and proportion of leg tarsal segments, is immensely diverse. In Drosophila melanogaster, adult legs have five tarsal segments. Accumulating evidence indicates that tarsal segments are formed progressively through dynamic changes in the expression of transcription factor genes, such as Bar genes, during development. In this study, to examine further the basis of progressive tarsal patterning, the precise expression pattern and function of several transcription factor genes were investigated in relation to the temporal regulation of Bar expression. The results indicate that nubbin is expressed over a broad region at early stages but gradually disappears from the middle of the tarsal region. This causes the progressive expansion of rotund expression, which in turn progressively represses Bar expression, leading to the formation of the tarsal segment 3. The region corresponding to the tarsal segment 4 is formed when apterous expression is initiated, which renders Bar expression refractory to rotund. In addition, the tarsal segment 2 appears to be derived from the region that expresses Bar at a very early stage. Cessation of Bar expression in this region requires the function of spineless, which also regulates rotund expression. These findings indicate that the temporally dynamic regulatory interaction of these transcription factor genes is the fundamental basis of the progressive patterning of the tarsal region.

Joint morphology in the insect leg: evolutionary history inferred from Notch loss-of-function phenotypes in Drosophila.

Joints permit efficient locomotion, especially among animals with a rigid skeleton. Joint morphologies vary in the body of individual animals, and the shapes of homologous joints often differ across species. The diverse locomotive behaviors of animals are based, in part, on the developmental and evolutionary history of joint morphogenesis. We showed previously that strictly coordinated cell-differentiation and cell-movement events within the epidermis sculpt the interlocking ball-and-socket joints in the adult Drosophila tarsus (distal leg). Here, we show that the tarsal joints of various insect species can be classified into three types: ball-and-socket, side-by-side and uniform. The last two probably result from joint formation without the cell-differentiation step, the cell-movement step, or both. Similar morphological variations were observed in Drosophila legs when Notch function was temporarily blocked during joint formation, implying that the independent acquisition of cell differentiation and cell movement underlay the elaboration of tarsal joint morphologies during insect evolution. These results provide a framework for understanding how the seemingly complex morphology of the interlocking joint could have developed during evolution by the addition of simple developmental modules: cell differentiation and cell movement.

Dynamic shape changes of ECM-producing cells drive morphogenesis of ball-and-socket joints in the fly leg.

Animal body shape is framed by the skeleton, which is composed of extracellular matrix (ECM). Although how the body plan manifests in skeletal morphology has been studied intensively, cellular mechanisms that directly control skeletal ECM morphology remain elusive. In particular, how dynamic behaviors of ECM-secreting cells, such as shape changes and movements, contribute to ECM morphogenesis is unclear. Strict control of ECM morphology is crucial in the joints, where opposing sides of the skeleton must have precisely reciprocal shapes to fit each other. Here we found that, in the development of ball-and-socket joints in the Drosophila leg, the two sides of ECM form sequentially. We show that distinct cell populations produce the 'ball' and the 'socket', and that these cells undergo extensive shape changes while depositing ECM. We propose that shape changes of ECM-producing cells enable the sequential ECM formation to allow the morphological coupling of adjacent components. Our results highlight the importance of dynamic cell behaviors in precise shaping of skeletal ECM architecture.

GATAe-dependent and -independent expressions of genes in the differentiated endodermal midgut of Drosophila.

Two sequentially-expressed GATA factor genes, serpent (srp) and GATAe, are essential for development of the Drosophila endoderm. The earliest endodermal GATA gene, srp, has been thought to specify the endodermal fate, activating the second GATA gene GATAe, and the latter continues to be expressed in the endodermal midgut throughout life. Previously, we proposed that GATAe establishes and maintains the state of terminal differentiation of the midgut, since some functional genes in the midgut require GATAe activity for their expression. To obtain further evidence of the role of GATAe, we searched for additional genes that are expressed specifically in the midgut in late stages, and examined responses of a total of selected 15 genes to the depletion and overexpression of GATAe. Ten of the 15 genes failed to be expressed in the embryo deficient for GATAe activity, but, the other five genes did not require GATAe. Instead, srp is required for activating the five genes. These observations indicate that GATAe activates a major subset of genes in the midgut, and some other pathway(s) downstream of srp activates other genes.

Fate determination of Drosophila leg distal regions by trachealess and tango through repression and stimulation, respectively, of Bar homeobox gene expression in the future pretarsus and tarsus.

During tissue patterning, developing fields may be subdivided into several non-overlapping domains by region-specific expression of transcription factors. In Drosophila leg development, the most distal segments, the pretarsus and tarsal segment 5 (ta5), are precisely specified by interactions between tarsus homeobox genes (BarH1 and BarH2) and pretarsus homeobox genes (aristaless, clawless, and Lim1). Here, we demonstrate that trachealess and tango, both encoding bHLH-PAS proteins that are required for the formation of the embryonic tracheal system, are essential for forming two adjacent distal segments of the leg. trachealess is expressed in the pretarsus and ta5, and the concerted action of trachealess and tango seems to modulate the activity of homeobox gene regulatory loops by repressing Bar in the pretarsus and activating Bar in ta5.

Temporal regulation of late expression of Bar homeobox genes during Drosophila leg development by Spineless, a homolog of the mammalian dioxin receptor.

The spatial and temporal regulation of genes encoding transcription factors is essential for the proper development of multicellular organisms. In Drosophila leg development, the distal-most tarsus (ta5) is specified by the strong expression of a pair of Bar homeobox genes in late third instar. This expression is regulated under the control of the ta5 enhancer activated by Bar. No activation of the ta5 enhancer, however, occurs in early third instar when considerable Bar is produced. The ta5 enhancer was comprised of a basal enhancer required for driving Bar expression and a negative regulatory motif serving as a binding site for the heterodimer of Spineless and Tango, homologs of the mammalian dioxin receptor and aryl hydrocarbon nuclear translocator, respectively. The spineless and tango were essential for suppressing the basal enhancer activation in early third instar. The spineless was transiently expressed in early third instar in the Bar expression domain. ta5 Bar expression may thus be temporally regulated through transient inhibition of premature activation of the basal enhancer via specific binding of the Spineless/Tango heterodimer to the negative regulatory motif in early third instar and subsequent release from the inhibition due to the disappearance of spineless expression at later stages.