Loss of thrombospondin reveals a possible role for the extracellular matrix in chordotonal cap cell elongation

Abstract

In the Drosophila larva, major proprioceptive input is provided to the brain by subepidermal stretch receptors called chordotonal organs (ChO). Similarly to the body wall muscle that needs to be attached on both of its sides to the larval exoskeleton in order to generate movement, the sensory unit of a ChO must be stably anchored to the cuticle on both of its sides in order to sense the relative displacement of body parts. Through an RNAi screen we have identified thrombospondin (Tsp), a secreted calcium binding glycoprotein, as a critical component in the anchoring of ChOs to the cuticle. We show that the Tsp protein starts to accumulate in the extracellular matrix (ECM) surrounding the ChO attachment cells towards the end of embryogenesis and that it becomes highly concentrated at the attachment junction during larval stages. In the absence of Tsp, the ChO's accessory cells fail to form a stable junction with their epidermal attachment cells and organ integrity is not maintained. Tsp is a known player in the establishment of the myotendinous junctions in both invertebrates and vertebrates. Thus, our findings extend the known similarities between muscle-attachment and ChO-attachment cells. In addition to its role in establishing the ChO attachment junctions, Tsp was found to affect ligament cell migration and cap cell elongation. Most interestingly, the Tsp protein was found to decorate the ChO cap cells along their entire length, suggesting that the elongated cap cells are supported by the ECM to which they attach via integrin-based, Tsp-dependent, adhesion plaques. The ECM enwrapping the cap cells is probably important for keeping the cap cells fasciculate and may also provide mechanical support that allows the extremely elongated cells to maintain tension.

An RNAi Screen Identifies New Genes Required for Normal Morphogenesis of Larval Chordotonal Organs

“…In wildtype larvae, the cap cells are stretched between the scolopale cells and the CA cells. During larval growth, the CA cells grow dramatically, extending numerous tubulin-rich extensions and forming a wide integrin-rich junction with the attached cap cells ( Halachmi et al 2016 ; Greenblatt Ben-El et al 2017 ). These morphological changes are likely required for adjusting the ability of the CA cells to anchor the cap cells and remain attached to the cuticle under conditions of increasing mechanical stresses.…”

Building functional units of movement-generation and movement-sensation in the embryo

“…Similarly to the musculoskeletal system the three components of the proprioceptive system are tightly linked through specialized junctional components required for the maintenance of organ integrity and function. Many of the proteins characterizing the myotendinuous junction (MTJ), including specific adhesion receptors and extracellular matrix components, are shared by the ChO junction (Greenblatt Ben- El et al, 2017). As discussed below, some of the molecular mechanisms involved in the assembly of the musculoskeletal system are also involved in the assembly of functional proprioceptors.…”

“…Interestingly, during larval stages Tsp does not appear to be maintained in the MTJ and is detected in vesicular-like particles in the muscle cytoplasm, possibly involved in protein translocation (Volk, T. personal observations). Tsp also plays an essential role in the attachment of ChO to their attachment cells and is required for proper localization of integrin in the cap cells (Greenblatt Ben- El et al, 2017). Although the stop signal for migrating ChO ligament or cap cells has not been identified, we have evidence demonstrating that the ligament-attachment cell is critical for stopping the migrating ligament cells of the LCh5 organ and later on for stopping the migration of the ventral VChB cap cell (Halachmi et al, 2016;Hassan et al, unpublished data).…”

“…Although the stop signal for migrating ChO ligament or cap cells has not been identified, we have evidence demonstrating that the ligament-attachment cell is critical for stopping the migrating ligament cells of the LCh5 organ and later on for stopping the migration of the ventral VChB cap cell (Halachmi et al, 2016;Hassan et al, unpublished data). In addition, phenotypic analysis of tsp mutant embryos shows defects in ChO migration and a failure of the ventral ChO (VChB) to stall when they reach the ventral lateral transverse tendon cells (Greenblatt Ben- El et al, 2017).…”

“…Both types of cells are defined by the expression of the transcription factor Stripe, they both require the bHLH transcription factor Delilah (Dei) for terminal differentiation, and both exhibit an elaborate MT-network. The connection between the ChO's cap and ligament cells to their corresponding epidermal attachment cells involves junctional elements similar to those used to connect muscles to tendons, such as integrin adhesion receptors, Shot and Tsp (Greenblatt Ben-El at al., 2017). This similarity reflects their function in force transmission.…”