Transcriptomic and Genetic Analyses Identify the Krüppel-Like Factor Dar1 as a New Regulator of Tube-Shaped Long Tendon Development

Abstract

To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During the leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. We provide for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, we identified the Krüppel-like factor Dar1 as a critical actor of leg tendon development. Specifically expressed in the leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. Our findings show that Dar1 acts downstream of Stripe and is required to set up the correct number of tendon progenitors.

Developmental origin of tendon diversity in Drosophila melanogaster

“…In the legs, the main muscles consist of multiple fibers that are attached to a long internal tendon on one side and to a single MAS localized beneath the leg cuticle on the other side ( Soler et al, 2004 ). Both types of tendon (long and cuticular) express sr , and the mechanisms that induce their expression are gradually being identified ( Laddada et al, 2019 ; Laurichesse et al, 2021 ). From the third instar stage of larval development and the first hours of pupae metamorphosis, sr starts to be expressed in seven clusters of epithelial cells within the presumptive joints that will form the future connections between leg segments ( Soler et al, 2004 ; Laddada et al, 2019 ) ( Figure 1C ).…”

“…In the leg disc, tendon cell clusters adopt a particular shape during metamorphosis. Each initial cluster of Sr+ cells undergoes a progressive invagination followed by a collective migration leading to the formation of a long polarized tube-like structure surrounding a central lumen ( Soler et al, 2004 ; Laddada et al, 2019 ; Laurichesse et al, 2021 ) ( Figure 2C ). The initial invagination of leg tendon precursors appears to occur independent of sr activity.…”

“…However, the reduction in sr expression precludes the elongation of the tendon, suggesting that leg disc sr is required to trigger a differentiation program that allows the collective migration of these cells ( Laddada et al, 2019 ). Interestingly, the expression of the Krüppel-like factor dar1 is restricted to the appendicular long tendons and is not found in other tendon precursors that do not form long internal structures (e.g., tendons of the flight muscles or larval muscles) ( Laurichesse et al, 2021 ). Moreover, Dar1 acts downstream of sr and participates in the cellular events required for tendon elongation such as the formation of actin-rich filopodia at the basal membrane of migrating cells ( Figure 2C ).…”

“…Moreover, Dar1 acts downstream of sr and participates in the cellular events required for tendon elongation such as the formation of actin-rich filopodia at the basal membrane of migrating cells ( Figure 2C ). Consequently, dar1 knockdown leads to a shortening of the leg tendons and a reduction in the number of Sr+ cells though it is not required cell-autonomously to induce sr expression, suggesting that tendon elongation may indirectly participate in the recruitment of new Sr+ tendon cells from the epithelium ( Laurichesse et al, 2021 ). Strikingly, throughout this process of long tendon development, subpopulations of leg muscle precursors are organized around each cluster of tendon precursors and remain firmly associated with them as the tendons elongate ( Soler et al, 2004 ; Maqbool et al, 2006 ).…”

Amalgam plays a dual role in controlling the number of leg muscle progenitors and regulating their interactions with developing tendon

“…We previously reported transcriptional signatures of leg tendon precursor cells (Laurichesse et al 2021) at the developmental time point of 0 h after pupae formation (APF) when subpopulations of myoblasts localize around clusters of tendon precursors. Here, we analyzed the transcriptome of leg disc myoblasts at the same time of development to identify potential interacting pairs.…”