Odd-skipped and Stripe act downstream of Notch to promote the morphogenesis of long appendicular tendons in Drosophila

Abstract

Multiple tissue interactions take place during the development of the limb musculoskeletal system. While appendicular myogenesis has been extensively studied, development of connective tissue associated with muscles has received less attention. In the developing Drosophila leg, tendon-like connective tissue arises from clusters of epithelial cells that invaginate into the leg cavity and then elongate to form internal tube-shape structures along which muscle precursors are distributed. Here we show that stripe-positive appendicular precursors of tendon-like connective tissue are set up among intersegmental leg joint cells expressing odd-skipped genes, and that Notch signaling is necessary and locally sufficient to trigger stripe expression. This study also finds that odd-skipped genes and stripe are both required downstream of Notch to promote morphogenesis of tube-shaped internal tendons of the leg.

Keywords: Drosophila leg; Muscle development; Notch; Odd-skipped; Tendon; Tubulogenesis.

Fig. 1.

Tendon precursors arise from Odd-skipped positive cells. Confocal images of leg disc tendons are revealed by Stripe-Gal4>UAS-mCherryNLS (magenta) and segmental true joints by Odd^RK11lacZ expression (green). (A–C) Selected optical sections of L3 disc, Sr>mCherry expression is detected among rings of Odd-expressing cells of pre-tarsus/T5 joint (ring 1, arrowheads) and femur/tibia dorsal junction (ring 3, asterisks). Remarkably, at pre-tarsus/T5 junction, Sr>GFP-positive cells form a ring surrounding a lumen (arrowheads) prefiguring the formation of the long tendon of the tarsi. Note that only cells at the surface are visible on these sections and no Sr>mCherry cells are detected along the T1/tibia true joint at this stage (ring 2). (D–F) Leg discs at the beginning of pupation, long tendons of the tarsi have grown deeply into the leg cavity (arrowheads). The number of Sr>mCherry cells in the dorsal femur have increased (asterisks), some of these cells expressed no or very low levels of Odd-lacZ (dashed outlined areas). Note the presence of a few Sr>mCherry cells along the T1/tibia joint (ring 2). (G–I) On everting leg disc at 3 h after pupae formation (APF), all tendon precursors were specified and co-expressed Sr>mCherry and Odd-lacZ. Sr>mCherry cells (arrowheads), arising from pre-tarsus/T5 joint (ring 1 in G), have deeply invaginated to form the long internal tendon of the tarsi. Tendon precursor (asterisks) associated with the femur/tibia joint (ring 3 in G) is beginning to invaginate. Note the nascent tendon precursor expressing Sr>mCherry (arrows) along the tibia/T1 joint (ring 2 in G).

Fig. 2.

Notch pathway activity in tendon precursors. Expression of Notch (cyan), Sr-lacZ (magenta) and Gbe-Su(H)-GFP (green) in leg discs. (A–D) Early L3, Notch (arrow in A) is first detected at the apical surface of Sr-LacZ cells corresponding to the future tarsi long tendon (B); these cells express Gbe-Su(H)-GFP, Notch pathway activity reporter (arrows in C and D). (E–H) From mid L3, Sr-lacZ accumulates in the developing tendon of the dorsal femur following the expression of Notch protein and Gbe-Su(H)-GFP (arrowheads). (I–L) 3 h APF, in selected optical sections, Sr-LacZ cells invaginate in the dorsal femur to form long tendon accumulating Notch proteins at the apical surface (arrowheads in I and J); these cells show Notch-positive activity (dashed outlined areas in K and L). Note the appearance of later tendon precursors in other segments also accumulating Gbe-Su(H)-GFP expression (arrows in J–L).

Fig. 3.

Notch signaling is required for Stripe expression. (A–F) Notch (cyan), Sr-lacZ expression (green) and UAS-mCherryCAAX (magenta) in R10H12-Gal4 (A–C) and R10H12-Gal4 >UAS-Notch-RNAi (D–F) leg discs at 0 h APF. (A–C) Notch is detected in invaginating tendons: selected optical sections show the tube-like shape formed by the long tendon in tarsi (double-headed arrows) and lumen aperture of the dorsal femur tendon (arrows). R10H12-gal4 pattern overlaps with the tendon precursor in dorsal femur, but not with the long tendon in tarsi (merge in C). (D–F) UAS Notch-RNAi expression strongly repressed Notch expression in R10H12-gal4 pattern (D and merge in F), including dorsal femur where Sr-lacZ expression is lost (arrows in E and F).

Fig. 4.

Forced Notch pathway activation causes Sr-LacZ ectopic expression and ectopic tendon formation. (A–C) Control leg disc at 0 h APF, Sr-lacZ positive cells in dorsal femur (arrow in A), surround lumen developing tendon revealed by FasIII apical accumulation (arrow in B), merged channels shown in C. (D–F) In Dpp-gal4>UAS-N^intra leg disc, Notch pathway activation leads to ectopic SrlacZ expression in a cluster of cells in dorsal femur (asterisks in D and F) able to invaginate (arrowhead in E). Note that more Sr-lacZ cells are recruited to form endogenous dorsal femur tendon (brackets in D and F) compared to control (A), and so these cells form a wider lumen aperture as revealed by FasIII staining (arrows in E and F). (G–I) Clone of cells expressing N^intra in L3 leg disc, marked by GFP expression (green in I). Sr-lacZ marked original tendon precursors (arrows) and is also induced in one of the clones in dorsal femur invaginating and starting to form an internal tube (arrowheads).

Fig. 5.

Odd-skipped and Stripe interact to form long internal tendons. (A–F) R10H12-Gal4>UAS-mCherryCAAX (magenta) leg discs at 5 h APF immunostained with anti-Sr (green) and anti-FasIII (cyan). (A) Control leg disc and (B) leg disc expressing UAS-BowlRNAi. (C,E) Higher magnifications from A showing Sr-expressing cells forming a long internal tube that elongates into the dorsal femur cavity. (D,F) Higher magnifications from B; number of cells expressing Sr is significantly reduced after Bowl-RNAi expression (D), remaining Sr-positive cells can still invaginate to form a tube reduced in size (compare F with E). (G) Box-plot diagram comparing number of Sr-positive cells in dorsal femur tendon in control and after expression of UAS-BowlRNAi. (H) R10H12-Gal4;Odd-lacZ>UAS-SrRNAi leg discs at 5 h APF immunostained with anti-lacZ (magenta) and anti-FasIII (cyan) and anti-Sr (green). (I) Single channel showing complete absence of Sr protein in dorsal femur where R10H12-Gal4 drives UAS-SrRNAi expression (arrow). Conversely, Sr is still expressed in other tendons (asterisks). (J,K) High magnification of dorsal femur from (H) for Odd-lacZ and FasIII channels respectively. Odd-lacZ expression is maintained in absence of Sr, FasIII accumulation at disc surface indicates that Odd-lacZ+ cells can trigger constriction of the disc epithelium but cannot form an internal tube. (M) In our model, Notch induces Odd in presumptive true joints leading to epithelium folding, Notch/Odd conjointly with unknown signal (X) trigger Sr expression responsible for tendon elongation.