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Fig. 4.
The fibronectin (FN) matrix is defective next to cells with compromisedaqp3bexpression within the DMZ. Gastrula embryos (stage 10.5) that had been co-injected with MOs and membrane-bound mem-EGFP tracer RNA (green) were hemisected sagittally through the dorsal blastopore lip, stained with FN antibodies (red), and evaluated by confocal microscopy. (A) In a control MO1-injected gastrula embryo, the FN matrix extended along the inner surface of the blastocoel roof and into the dorsal and ventral marginal zones. (B-H) Control MO-injected embryos had an uninterrupted FN matrix. The dashed box in the lower magnification image (B) indicates the area shown in (C, F) from a different optical plane. Continuous FN matrix was also present in embryos injected with control MO2 (D, G) and both control MOs (E, H). The green channel (mem-EGFP) was omitted in the lower panels to clearly show the extent of the FN matrix (red), which was continuous between non-involuted ectoderm and involuted mesendoderm. (I-O) aqp3b morphants showed interruption of the FN matrix wherever aqp3b MOs were present (marked by green mem-EGFP tracer). The dashed box in (I) shows an equivalent area as in (J, M). In the area sandwiched between ectoderm and mesendoderm, the fibrillar FN matrix is highly disrupted by the presence of aqp3b MO1 (I, J, M), aqp3b MO2 (K, N), and synergistically in embryos injected with lower amounts of both aqp3b MOs (L, O). DMZ, dorsal marginal zone; VMZ, ventral marginal zone; BCR, blastocoel roof; DLB, dorsal blastopore lip; e, ectoderm; me, mesendoderm; bc, blastocoel cavity; the dotted lines demarcate the floor of the blastocoel. Scale bars: 50 um. |
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Fig. 5.
Cells with disruptedaqp3black a continuous fibrillar FN matrix across the face of the ectoderm. Confocal image stacks of hemisected embryos were rotated to show the surface of ectoderm cells (B-E, G-J). Control MO1-injected embryos (A-E) showed an extensive fibrillar FN matrix across the face of the ectoderm in Brachet's cleft (B, C) and along the BCR (D, E). aqp3b MO1-injected embryos (F-J) were nearly devoid of fibrillar FN matrix across the face of the ectoderm in Brachet's cleft (G, H), but did possess FN matrix lining the face of the BCR (I, J). The boxes in (A) and (F) indicate the areas that were used for the rotated projections (dashed box = BCR: B, C, G, H, and solid box = Brachet's cleft: D, E, I, J). The green channel was removed in (C, E, H, J). The dotted lines delineate the floor of the blastocoel. Scale bars: (A, F) 100 um; (B-E, G-J) 10 um. |
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Fig. 6.
Cells protruding past the defective tissue boundary. (A, B, E, F) Control MO and mem-EGFP (green) injected cells showed an intact boundary between ectoderm and mesendoderm cells, which was respected by all cells. (FN matrix is labeled red.) (C, D, G, H) Cells injected with aqp3b MOs lacked FN matrix, which allowed abnormal behavior of cells at the tissue border. Cells failed to respect the tissue boundary, forming protrusions that extend past the border between ectoderm and mesendoderm (arrows). Scale bars: 30 um. |
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Fig. 7.
Embryos that receivedaqp3bMO localized non-fibrillar FN matrix around all cells. Embryos co-injected with both aqp3b MOs (4 ng each) lacked a continuous fibrillar fibronectin (FN) matrix (C, D) but exhibited the same level of pericellular FN matrix (arrowheads) as control MOs-injected cells (A, B), suggesting that the presence of non-fibrillar FN was not affected by the absence of Aqp3b. Asterisks in (C-F) mark cells that did not receive aqp3b MOs in otherwise aqp3b MOs-injected embryos. These cells had adjacent fibrillar FN matrix (white arrows), unlike neighboring cells, which received aqp3b MOs and where the fibrillar FN matrix was lacking. Scale bars: 10 um.
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