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Fig. 3. Adprhl1 morpholino disrupts myofibril assembly in the forming ventricle. Comparison of dissected hearts from tadpoles injected with Adprhl1-e2i2MO (A, B, E, F) into d-2/4 blastomeres versus those containing control morpholino (C, D, G, H). Phalloidin stain of actin filaments (red) and immunocytochemistry for myosin heavy chain (green) is presented (+DAPI nuclei-blue-F, H). S. Fig. 6 contains additional data, including higher magnification images for stage 33, 37 and stage 40 ventricles. A-D: At the onset of cardiac looping, stage 33, left lateral view of heart tubes. (apex-white arrowheads). E-H: After outgrowth of the ventricle, stage 40 hearts (E, G) oriented with anterior surface upwards, showing myofibril patterns (arrowheads) in the ventricle wall (F, H). Scale bars = 100 μm (A, C, E, G). Scale bars = 10 μm (F, H). I: Cell number and size measurements for the hearts depicted here and S. Fig. 6. At stage 33, cardiac looping is more advanced in the control heart but cell surface area is comparable to the morphant. At stage 37 and 40, fewer cells are present on the surface of smaller morphant ventricles. J-N: Illustrations showing normal morphology and myofibril patterns in the forming ventricle. J: Wildtype stage 33 heart tube, depicting cardiomyocytes on the left side that elongate to form a rosette structure, an early cell movement observed during looping morphogenesis that is absent from morphants. K: A stage 40 heart ventricle, with contours (grey lines) to represent circumference-axes running from base to apex (from inner to outer curvature). Alignment of myofibrils is described relative to these axes. L-N: Simplified arrangement of myofibrils at three locations within the ventricle chamber. Myofibrils near the base (inner curvature-L) orient parallel to the axes. Conversely, around the apex, the predominant orientation of larger myofibrils extends perpendicular to the axes (M), at least for cardiomyocytes on the outer (apical) surface of the chamber. At deeper positions towards the lumen, myofibrils of trabecular (Tr) cardiomyocytes align parallel to the axes (N). There is no abrupt boundary between cardiomyocytes that contain perpendicular or parallel-oriented myofibrils. Moreover, the extent of the perpendicular cells is greater on the anterior ventricle wall (left sided origin) and lesser on the posterior wall (right origin). |
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Fig. 6.
Adprhl1 morpholino disrupts myofibril assembly in the forming ventricle.A-D, I-L, Q-T: Dissected hearts from tadpoles injected with Adprhl1-e2i2MO into D-2/4 blastomeres and analyzed at stage 33 (A-D), stage 37 (I-L) and stage 40 (Q-T). E-H, M-P, U-X: Hearts after control morpholino injection into D-2/4 blastomeres. Phalloidin stain of actin filaments (red) and immunocytochemistry for myosin heavy chain (green) are presented. A-H: At the onset of cardiac looping, stage 33, left lateral view of heart tubes (A, B, E, F). Squares denote the position of 105 μm images (C, D, G, H) in the presumptive ventricle region (apex-white arrowheads). Knockdown of adprhl1 prevents the rearrangement of cortical actin and delays myosin production in the ventricle, thus inhibiting formation of striated myofibrils (coloured arrowheads). Additionally, elongation of cardiomyocytes located near the apex fails to occur. I-P: During ventricle outgrowth, stage 37 hearts (I, M) oriented with anterior surface upwards, showing myofibril patterns in the ventricle wall (J-L, N-P). Myofibrils that eventually form after adprhl1 knockdown are disarrayed (white arrowheads) whereas control myofibrils near the apex extend in a perpendicular direction (coloured arrowheads). Q-X: At stage 40, comparable series of hearts (Q, U) and ventricle images (R-T, V-X). Despite increased synthesis, no recovery of myofibril order and direction occurs with the adprhl1 morpholino. Arrowheads mark equivalent sarcomere positions in the paired panels. Scale bars = 100 μm (A, E, I, M, Q, U). Scale bars = 10 μm (all others). |
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Fig. 7.
Adprhl1 morpholino affects myofibrils but not cellular membrane structure.A-G: A stage 37 heart, dissected after co-injection of Adprhl1-e2i2MO with membrane-tagged lyn-mCherry RNA into D-2/4 blastomeres. H-N: Heart after control morpholino and lyn-mCherry RNA injection into D-2/4 blastomeres. The anterior surface of the hearts (A, B, H, I) lies upwards and magnified 134 μm images (C-G, J-N) show myofibril and membrane patterns in the ventricle wall. Phalloidin actin (magenta-A, C, H, J), membrane-tagged mCherry (red-B, D, I, K), anti-myosin heavy chain (green-E, L), DAPI (F, M) stains, plus RGB-channel merge (G, N). Panels show reduced myofibril content (arrowheads) in the forming ventricle upon adprhl1 knockdown but no clear change in cellular membrane shape or vesicle structure. Scale bars = 100 μm (A, H). Scale bars = 10 μm (C-G, J-N). White arrowheads, short, splayed myofibrils in morphant. |
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Fig. 11.
Absence of a signal for endogenous Adprhl1 protein in tadpole hearts. Despite the mRNA for adprhl1 being an abundant molecule in embryonic hearts, endogenous levels of Adprhl1 protein are low and are not readily detected by immunocytochemistry. Scarce fluorescent dots observed using the Adprhl1 antibody can be detected in superficial optical sections of all embryonic tissues and are not specific. A-C: Cardiomyocytes in the ventricular region of a looping stage 34 heart tube. A: Anti-Adprhl1, using a far-red channel, fluorescent secondary antibody. B: Phalloidin, anti-myosin heavy chain and DAPI. C: Merge of four channels. D-F: Similar sequence of images of cardiomyocytes from the apex of the ventricle at stage 40. G-I: Stage 44 ventricular cardiomyocytes, this time using a different green channel, fluorescent secondary antibody to attempt Adprhl1 detection. Scale bar (A) = 10 μm and applies to all images. |
