Figure 1.
Annexin A6 transcript distribution in the developing chick embryo.
(A,C,E,G,I) Whole-mount in situ hybridization for Annexin A6 followed by indicated transverse sections (B,D,F,H,J) shown for specific developmental stages (A,B: 4 ss; C,D: 5 ss; E,F: 6 ss; G,H: 7 ss; I–J: 8 ss). (K) Representative midbrain transverse section taken from an 8 ss embryo that has undergone whole-mount in situ hybridization for Annexin A6 followed by sectioning and immunohistochemistry for the migratory neural crest cell marker HNK-1 (red). Arrowheads in (F,H,J,K) indicate Annexin A6 mRNA in migratory neural crest cells. Scale bars in (A) and (B) are 200 and 50 µm, respectively, and are applicable to respective image types.
Figure 2.
A 5 base pair mismatch Annexin A6 control MO does not affect chick midbrain neural crest cell emigration in vivo.
(A) Immunoblots for Annexin A6 and β-actin showing reduced levels of Annexin A6 protein upon Annexin A6 MO treatment, with no change observed upon control MO treatment. (B,D) Whole-mount in situ hybridization followed by indicated transverse sections for Sox10 (C) and Snail2 (E), respectively, after 8 hour incubation following treatment with control MO. (F) Representative transverse section taken from an embryo treated with control MO for 8 hours followed by immunohistochemistry for HNK-1 (green). No change is noted in whole-mount images or sections with respect to neural crest cell emigration or migration. In all experiments, the right side of the embryo is electroporated, as indicated by the lissamine (red) fluorescence of the MO in the transverse sections (C,E) and/or in the inset images of each whole-mount (B,D). Scale bar in (B) is 50 µm and applicable to all whole-mount and section images. MO, red; DAPI, blue.
Figure 3.
MO-mediated depletion of Annexin A6 from the developing neural crest cell population of the chick midbrain diminishes the size of the premigratory and migratory neural crest cell domains.
(A,C) Whole-mount in situ hybridization followed by indicated transverse sections for Sox10 (B) and Snail2 (D), respectively, after 8 hour incubation following treatment with Annexin A6 MO. (E) Representative transverse section taken from an embryo treated with Annexin A6 MO for 8 hours followed by immunohistochemistry for HNK-1 (green). (F,H) Whole-mount in situ hybridization followed by indicated transverse section (G,I) for Sox10 after 3 and 20 hour incubation following treatment with Annexin A6 MO, respectively. (J) Representative transverse section taken from an embryo treated with Annexin A6 MO for 3.5 hours followed by Snail2 whole-mount in situ hybridization. Arrows in (B–J) indicate the migratory or premigratory neural crest cell domain. In all experiments, the right side of the embryo is electroporated, as indicated by the lissamine (red) fluorescence of the MO in the transverse sections (B,D,E,G,I,J) and/or in the inset images of each whole-mount (A,C,F,H). (K) Graphical representation of changes in the premigratory (Snail2) and migratory (Sox10) neural crest cell populations upon depletion of Annexin A6. Scale bar in (A) is 50 µm and applicable to all whole-mount and section images except for that shown in (I) where the scale bar is also 50 µm. MO, red; DAPI, blue.
Figure 4.
Depletion of Annexin A6 does not alter cell proliferation or cell death in the chick embryonic neural tube or migratory neural crest cell population.
(A–D) Electroporation of control (A,C) or Annexin A6 (B,D) MO, followed by 8 hour incubation, transverse sectioning, and processing for phospho-histone H3 immunohistochemistry (A,B, PH3, green) or TUNEL (C,D, green) (representative sections are shown). Arrowheads indicate PH3-positive (A,B) or TUNEL-positive (C,D) nuclei, with a similar distribution observed in the neural tube and in migratory neural crest cells in the presence of either MO and with that found on the contralateral control side of the embryo. In all experiments, the right side of the embryo is electroporated with the MO, as indicated by the lissamine (red) fluorescence of the MO in the sections. Scale bar in (A) is 50 µm and applicable to all images. MO, red; DAPI, blue.
Figure 5.
MO-mediated depletion of Annexin A6 leads to retention of molecular markers of adherens junctions.
(A–D) Representative transverse section taken through the midbrain of an embryo electroporated with Annexin A6 MO (red) after 6 (A–C) and 5 (D) hours of incubation and processing by immunohistochemistry for Claudin-1 (A), Cingulin (B), Cadherin6B (C), and N-cadherin (D) (all green), respectively. Arrows denote the maintenance of proteins on the electroporated side (right) of the neural tube. Scale bar in (A) is 50 µm and applicable to all images. MO, red; DAPI, blue.
Figure 6.
The pCIG control expression construct does not affect chick midbrain neural crest cell emigration in vivo.
(A) Immunoblots for Annexin A6 and β-actin showing increased levels of Annexin A6 protein upon pCIG-Annexin A6 treatment, with no change observed upon pCIG treatment. (B,D) Whole-mount in situ hybridization followed by indicated transverse sections (C,E) for Sox10 and Snail2, respectively, after 8 hour treatment with pCIG. (F) Representative transverse section taken from an embryo treated with pCIG for 8 hours followed by whole-mount immunohistochemistry for HNK-1 (red). No change is noted in whole-mount images or sections with respect to neural crest cell emigration or migration. In all experiments, the right side of the embryo is electroporated with pCIG, as indicated by the GFP (green) fluorescence in the transverse section (F) and/or in inset images of each whole-mount (B,D). Scale bar in (B) is 50 µm and applicable to all images. GFP, green; DAPI, blue.
Figure 7.
Overexpression of Annexin A6 in the developing neural crest cell population of the chick midbrain increases the size of the premigratory and migratory neural crest cell domains.
(A,C) Whole-mount in situ hybridization followed by indicated transverse sections for Sox10 (B) and Snail2 (D), respectively, after 8 hour incubation following treatment with pCIG-Annexin A6. (E) Representative transverse section taken from an embryo treated with pCIG-Annexin A6 for 8 hours followed by immunohistochemistry for HNK-1 (red). (F,H) Whole-mount in situ hybridization followed by indicated transverse section (G,I) for Sox10 after 4 and 20 hour incubation following treatment with pCIG-Annexin A6, respectively. (J) Representative transverse section taken from an embryo treated with pCIG-Annexin A6 for 6 hours followed by Snail2 whole-mount in situ hybridization. Arrows in (B–J) indicate the migratory or premigratory neural crest cell domain. In all experiments, the right side of the embryo is electroporated, as indicated by the GFP (green) fluorescence of the expression construct in (E) and/or in the inset images of each whole-mount (A,C,F,H). (K) Graphical representation of changes in the premigratory (Snail2) and migratory (Sox10) neural crest cell populations upon Annexin A6 overexpression. Scale bar in (A) is 50 µm and applicable to all whole-mount and section images except for that shown in (I) where the scale bar is also 50 µm. GFP, green; DAPI, blue.
Figure 8.
Annexin A6 overexpression does not alter cell death nor cell proliferation in the chick embryonic neural tube or migratory neural crest cell population.
(A–D) Electroporation of the pCIG control (A,C) or pCIG-Annexin A6 (B,D) constructs, followed by 8 hour incubation, transverse sectioning, and processing for phospho-histone H3 immunohistochemistry (A,B, PH3, red) or TUNEL (C,D, red) (representative sections are shown). Arrowheads indicate PH3-positive (A,B) or TUNEL-positive (C,D) nuclei, with a similar distribution in the neural tube and in migratory neural crest cells observed in the presence of either construct and with that found on the contralateral control side of the embryo. In all experiments, the right side of the embryo is electroporated with the construct, as indicated by the GFP (green) fluorescence in the sections. Scale bar in (A) is 50 µm and applicable to all images. GFP, green; DAPI, blue.
Figure 9.
Annexin A6 overexpression leads to precocious loss of molecular markers of adherens junctions.
(A–D) Representative transverse section taken through the midbrain of an embryo electroporated with pCIG-Annexin A6 (green) after 6 (A), 5 (B,C), and 4 (D) hours of incubation and processing by immunohistochemistry for Claudin-1 (A), Cingulin (B), Cadherin6B (C), and N-cadherin (D) (all red), respectively. Arrows denote the loss of proteins on the electroporated side (right) of the neural tube. Scale bar in (A) is 50 µm and applicable to all images. GFP, green; DAPI, blue.