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.

Here, we show for the first time that developmental cell death plays a critical role in the morphogenesis of multicellular proprioceptors in Drosophila. The most prominent multicellular proprioceptive organ in the fly larva, the pentascolopidial (LCh5) organ, consists of a cluster of five stretch-responsive sensory organs that are anchored to the cuticle via specialized attachment cells. Stable attachment of the organ to the cuticle is critical for its ability to perceive mechanical stimuli arising from muscle contractions and the resulting displacement of its attachment sites. We now show that five attachment cells are born within the LCh5 lineage, but three of them are rapidly eliminated, normally, by apoptosis. Strong genetic evidence attests to the existence of an autophagic gene-dependent safeguard mechanism that guarantees elimination of the unwanted cells upon perturbation of the apoptotic pathway prior to caspase liberation. The removal of the three superfluous cells guarantees the right ratio between the number of sensory organs and the number of attachment cells that anchor them to the cuticle. This accurate matching seems imperative for the attachment of cell growth and functionality and is thus vital for normal morphogenesis and functionality of the sensory organ.

Coordinated animal locomotion depends on the development of functional proprioceptors. While early cell-fate determination processes are well characterized, little is known about the terminal differentiation of cells within the proprioceptive lineage and the genetic networks that control them. In this work we describe a gene regulatory network consisting of three transcription factors–Prospero (Pros), D-Pax2, and Delilah (Dei)–that dictates two alternative differentiation programs within the proprioceptive lineage in Drosophila. We show that D-Pax2 and Pros control the differentiation of cap versus scolopale cells in the chordotonal organ lineage by, respectively, activating and repressing the transcription of dei. Normally, D-Pax2 activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. We further show that D-Pax2 and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer in which two D-Pax2- and three Pros-binding sites were identified experimentally. When this enhancer was removed from the fly genome, the cap- and ligament-specific expression of dei was lost, resulting in loss of chordotonal organ functionality and defective larval locomotion. Thus, coordinated larval locomotion depends on the activity of a dei enhancer that integrates both activating and repressive inputs for the generation of a functional proprioceptive organ.

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