PMC

Alary Muscles Act as Distal Anchors during Tubule Morphogenesis
(A–D) Ablation of the A3/A4, A4/A5, and A5/A6 alary muscles (MHC, red) perturbs anterior tubule architecture (A–C; control, D; Ct, green). Distal regions move further anteroventrally (A–C, arrows) and the kink moves distally (arrowheads); tip cells either fail to contact alary muscle targets (A′ and B′, arrow) or attach further anteriorly (C′, arrow).
(E–I) Manipulation of alary muscle anteroposterior identity does not affect tip cell choice. Three anterior-most alary muscle pairs (A1/A2–A3/A4) express Ubx (purple) but not Abd-A. Four posterior-most alary muscle pairs (A4/A5–A7/A8) express Abd-A (blue) but not Ubx. Asterisks indicate alary muscle targets. Ectopic Abd-A expression (tupGal4 in F; 24BGal4 in G and H) posteriorizes all alary muscles (E). 24BGal4>Abd-A embryos possess three additional alary muscle pairs, T1/T2–T3/A1 (G, arrowheads). 24BGal4>Abd-A^RNAi anteriorizes alary muscles (E). In all cases, tip cells (Ct, green) contact A3/A4 alary muscles (MHC, red) as normal (F′, H′, and I′, arrows).
(J–K) In Ubx (J) and dsparc (K) mutants, anterior tubules (Ct, brown, J; green, J′ and K) stall with their kinks in A2 (arrowheads) and tip cells bound to posterior alary muscles (MHC, red) at A5/6 (J′ and K′, arrows).
Scale bars represent 50 μm (A–D and F–K), 10 μm (A′ and B′), and 5 μm (C′, D′, F′, H′, and K′). See also .

A–D: Embryos were stained with antibodies for the Hox proteins Ubx or ABDA-A, and the accumulation of each protein was assessed in filleted samples. Representative alary muscles which were positive and negative for each Hox gene product are presented. Dorsal midline is to the top, anterior is to the left. The nuclei of the three most anterior alary muscle pairs were positive for the presence of Ubx (A), whereas more posterior alary muscles did not accumulate Ubx (B). Conversely, the anterior three alary pairs were negative for ABD-A (C), yet the posterior four alary pairs were positive for ABD-A (D). E: Summary of Hox gene expression and patterning in the alary muscles. Bar, 12.5μm.

Samples were stained for accumulation of MHC, and dissected as described above. Ventral views of filleted samples are shown. A: Wild-type, showing the normal pattern of seven pairs of alary muscles (arrows). B: Ubx mutants showed lack of a full complement of normally developed alary muscles, with the 2–3 most anterior pairs not observed. C: abd-A mutants showed a normal complement of alary muscles. D: Ubx and abd-A double-knockout mutants showed a large reduction in alary muscle number, with only occasional muscles observed (arrowhead). Bar, 100μm.

Stage 16 embryos ectopically expressing either Ubx (A) or abd-A (B) throughout the mesoderm were stained for accumulation of MHC and examined. In both cases, mutants showed an increased number of alary muscle pairs, from the seven normally observed in wild-type, to approximately ten (see text for details). The additional muscles were located in the more anterior region (arrowheads). Bar, 100μm.