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The proprioceptive chordotonal organs (ChO) of a fly larva respond to mechanical stimuli generated by muscle contractions and consequent deformations of the cuticle. The ability of the ChO to sense the relative displacement of its epidermal attachment sites likely depends on the correct mechanical properties of the accessory (cap and ligament) and attachment cells that connect the sensory unit (neuron and scolopale cell) to the cuticle. The genetic programs dictating the development of ChO cells with unique morphologies and mechanical properties are largely unknown. Here we describe an RNAi screen that focused on the ChO’s accessory and attachment cells and was performed in 2nd instar larvae to allow for phenotypic analysis of ChOs that had already experienced mechanical stresses during larval growth. Nearly one thousand strains carrying RNAi constructs targeting more than 500 candidate genes were screened for their effects on ChO morphogenesis. The screen identified 31 candidate genes whose knockdown within the ChO lineage disrupted various aspects of cell fate determination, cell differentiation, cellular morphogenesis and cell-cell attachment. Most interestingly, one phenotypic group consisted of genes that affected the response of specific ChO cell types to developmental organ stretching, leading to abnormal pattern of cell elongation. The ‘cell elongation’ group included the transcription factors Delilah and Stripe, implicating them for the first time in regulating the response of ChO cells to developmental stretching forces. Other genes found to affect the pattern of ChO cell elongation, such as αTub85E, β1Tub56D, Tbce, CCT8, mys, Rac1 and shot, represent putative effectors that link between cell-fate determinants and the realization of cell-specific mechanical properties.

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Highlights d The mechanical properties of the chordotonal organ are altered in Pericardin mutants d Loss of Pericardin drives a transition from compression to bending in the cap cell d Compressive strain within the cap cell is essential for proper sensing d The results can be interpreted using a simplified model of an elastic beam

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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.