A Tendon Cell Specific RNAi Screen Reveals Novel Candidates Essential for Muscle Tendon Interaction

<div><p>Tendons are fibrous connective tissue which connect muscles to the skeletal elements thus acting as passive transmitters of force during locomotion and provide appropriate body posture. Tendon-derived cues, albeit poorly understood, are necessary for proper muscle guidance and attachment during development. In the present study, we used dorsal longitudinal muscles of <i>Drosophila</i> and their tendon attachment sites to unravel the molecular nature of interactions between muscles and tendons. We performed a genetic screen using RNAi-mediated knockdown in tendon cells to find out molecular players involved in the formation and maintenance of myotendinous junction and found 21 candidates out of 2507 RNAi lines screened. Of these, 19 were novel molecules in context of myotendinous system. Integrin-βPS and Talin, picked as candidates in this screen, are known to play important role in the cell-cell interaction and myotendinous junction formation validating our screen. We have found candidates with enzymatic function, transcription activity, cell adhesion, protein folding and intracellular transport function. Tango1, an ER exit protein involved in collagen secretion was identified as a candidate molecule involved in the formation of myotendinous junction. Tango1 knockdown was found to affect development of muscle attachment sites and formation of myotendinous junction. Tango1 was also found to be involved in secretion of Viking (Collagen type IV) and BM-40 from hemocytes and fat cells.</p></div

Myotendinous junction formation fails in knockdown of CG33303, Tango1 and Tango4.

<p>(A) In Control myotendinous system at 24 h APF, cognate interaction of DLMs and their attachment site (tendons) is clearly visible (DLMs in green, attachment sites in red), filopodial extension of tendon cells are seen (white arrows). (B-D) In CG33303, <i>tango4</i> and <i>tango1</i> RNAi animals, DLMs are detached from their attachment site and show red speckles at their anterior ends (yellow arrows). Tendon cells do not show filopodial extension in these knockdown animals. (n = 4), white asterisk mark the target attachment site for DLMs.</p

Classification of RNAi candidates based on known/predicted function.

<p>* Embryonic lethal.</p><p>^# flight defect, rest were lethal at pupal stages.</p><p>Classification of RNAi candidates based on known/predicted function.</p

Myotendinous system phenotypes in tendon mediated RNAi knockdown at 36 h APF.

<p>Dorsal longitudinal muscles (DLMs) (MHC-tau-GFP, green) thoracic tendon cell clusters (<i>sr</i>-Gal4,UAS-myr mRFP, red) and myotendinous junction (β-PS integrin, blue) are shown at 36 h APF. (A) In control thoracic preparations, DLMs are attached to two tendon cluster anterior (dotted line) and posterior (yellow asterisk) and integrin accumulation is seen at the junction (anterior junction is shown with white arrow). Class I candidates (B-E) show severely affected tendon cell clusters (dotted line and yellow asterisk in B-D, white and yellow asterisk in E); DLMs are very small or absent. Class II candidates (F-I) show severe defect in tendon cells (dotted line and yellow asterisk in F, G, I and white and yellow asterisk in H,) similar to class I but, DLMs are small and attached to the posterior tendon cells (yellow asterisk) and show integrin accumulation at the anterior and posterior ends (anterior integrin accumulation is shown in white arrow). Class III candidates show developmental defect (J, K) wherein the thoracic preparations appear similar to an early staged preparation, tendon cells are marked with dotted line and yellow asterisk, larval templates are seen. Class IV candidates do not show any defect in myotendinous system (L) (n = 5).</p

Classification of candidates based on the knockdown phenotype with <i>sr</i>-Gal4.

<p>^<b>#</b> based on DRSC Integrative Ortholog Prediction Tool result.</p><p>Classification of candidates based on the knockdown phenotype with <i>sr</i>-Gal4.</p

Tendon cell development and differentiation affected in Tango1 knockdown.

<p>(A-A”) live control animal at 24 h APF shows DLMs (white asterisk in A, only two are visible in either hemisegment) and tendon cell clusters (a, b, c, d and yellow asterisk, dotted line shows the anterior target attachment site and yellow asterisk shows posterior attachment site for DLMs). Cognate interaction of DLMs and tendon cells are shown in A” (white arrows). (B-B”) live <i>sr</i>><i>tango1</i> RNAi animal at 24 h APF shows DLMs are not attached to its target tendon cell cluster at anterior end (dotted line, a) though it is attached to posterior cluster (yellow asterisk). Inset in B” shows the RFP speckles attached to the anterior end of DLMs. (C-C”‘) Tango1 staining in tendon cells is shown in control animal (in green, tendon cells are marked in red). (D-D”‘) Levels of Tango1 is low in <i>sr</i>><i>tango1</i> RNAi tendon cells (compare dotted area in D’ with C’ and inset in D” to C”). Muscles are marked in green in A-B”, Tango1 staining is labelled in green in c-D” and tendon cells are shown in red in all panels. (n = 4 for A and B, n = 7 for C and D)</p

Tango1 is required for Golgi organization and secretion of Collagen and BM-40 (SPARC).

<p>(A-B) Tango1 knockdown in hemocytes show accumulation of VkgGFP (B, compare with A). (C-D) Sparc is accumulated in hemocytes of Collagen-specific Tango1 knockdown L3 larvae (D) in comparison to hemocytes of control L3 larvae (C). (E-F) VkgGFP L3 larvae shows GFP expression throughout larval body (E), GFP expression is not seen throughout in L3 larvae of Collagen specific Tango1 knockdown, but high intensity GFP speckles are seen in fat body (F). (G-H’) In control larval flat preparations (VkgGFP), Collagen (marked by VkgGFP in F) is deposited in basement membrane; muscles are marked with Rhodamine labelled phalloidin (G’) as counterstain. In Collagen-specific Tango1 knockdown, levels of Collagen (marked by VkgGFP in H) are low, muscle are shown in H’. (I-L’) Golgi organization in Tango1 knockdown, hemocytes is affected shown by mannosidase-II GFP (J’, compare with I’) and GM130 (L’, compare with K’). I and J show Sparc staining, K and L show Vkg GFP staining in hemocytes. (M) Tango1 gene locus showing GS insertions, GS15095 and GS17108 and GS21664 at its 5’ end. (N-O) Hemocytes from L1 staged VkgGFP show no accumulation of VkgGFP inside cell (N), hemocytes from GS15095 homozygous L1 VkgGFP is accumulated inside cell (O). (P-R) BM-40 Sparc, an ECM protein is accumulated in the hemocytes of lethal GS insertion, GS15095 (Q) and GS17108 (R); compare with P. (S-U) Sparc is accumulated in fat cells of lethal GS insertions, GS15095 (P) and GS17108 (Q); compare with O. Note: n>20 for hemocytes, n = 5 for fat cells and body wall preparations. H and I are taken at Olympus stereozoom microscope; all others were imaged in Laser scanning confocal microscope.</p