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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.

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.

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.

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.

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.

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.