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First published online 14 November 2002
doi: 10.1242/jcs.00182

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Multiple connexins contribute to intercellular communication in the Xenopus embryo

Yosef Landesman^1,*, Friso R. Postma¹, Daniel A. Goodenough² and David L. Paul^1,

¹ Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
² Department of Cell Biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
^* Present address: GPC-Biotech Inc., 610 Lincoln Street, Waltham, MA 02451, USA

View larger version (32K): [in a new window]   Fig. 1. Cx38 RNA was symmetrically distributed in the 16-cell stage embryo. Embryos were separated into animal and vegetal parts and RNA was analyzed by northern blot. The Vg-1 antisense probe was used as a marker for transcripts from vegetal cells. Cx38 transcripts were equally abundant in animal and vegetal cells. Ribosomal RNA was used as a loading control (data not shown).

View larger version (46K): [in a new window]   Fig. 2. Antisense oligonucleotides deplete endogenous maternal Cx38 and depress GJIC in oocytes. Antisense (oligos 1-4) or sense oligonucleotides were injected into defolliculated oocytes. After 24 hours, total RNA was extracted from a subset of oocytes and northern blotted. Specific degradation of Cx38 RNA was seen for all four oligos. Vg-1 probe was used as a loading control. The remaining oocytes were paired with Cx43 cRNA-injected oocytes, and the development of electrical conductance was monitored [mean conductance levels in microsiemens (µS), SD and number of pairs in parenthesis are below the blot]. All antisense oligonucleotides tested reduced conductance to background levels.

View larger version (47K): [in a new window]   Fig. 3. Cx38 was not essential for either normal development or GJIC in the early embryo. Oocytes were injected with sense or antisense oligo-1, capacitated by host-transfer and fertilized. Dye transfer was assessed at the 32-64 cell stage. (A) Both sense and antisense injected oocytes developed into grossly normal swimming tadpoles. (B) Northern blotting confirmed the elimination of Cx38 mRNA from the embryos by oligo-1. (C-J) A mixture of neurobiotin (red) and fluorescein-dextran (green) was injected into one animal cell at the 32-64 cell stage. After fixation and sectioning, GJIC is evident (stars) in blastomeres that contain neurobiotin but not fluorescein-dextran. Bar (C), 200 µm.

View larger version (93K): [in a new window]   Fig. 4. Xenopus Cx31 and Cx43.4 are highly related to other connexins. (A) Xenopus Cx31 (accession AY057997) has 68-69% similarity to mouse, rat and human Cx31. (B) Xenopus Cx43.4 (accession AY057998) is most related to zebrafish Cx43.4 at 69% similarity but also shows 58-59% similarity to chicken, human, mouse and dog Cx45. Characteristic features of connexins, such as four predicted transmembrane domains (red bars) and triplets of cysteine residues (yellow C), are evident.

View larger version (38K): [in a new window]   Fig. 5. Four connexins were maternally expressed in Xenopus embryos. (A) RT-PCR from ovary, oocytes and early embryos distinguished between maternally and zygotically expressed Xenopus connexins. Cx30 was solely zygotic and Cx38 solely maternal. Cx31 and Cx43.4 were both maternal and zygotic. Histone H4 was used as an internal control. (B) RT-PCR analysis revealed that Cx43 RNA, but not Cx41, was expressed maternally in fertilized eggs and 64-cell stage embryos. RT-minus controls were utilized for all cDNA samples in A and B, but only one example is shown in each panel. (C) Steady-state levels of Cx38 and Cx43.4 RNA are compared by northern blots. Fibronectin (FN) levels are provided as a loading control. In all three panels oocytes samples were obtained from defolliculated oocytes and ovary samples from pieces of ovary containing follicular oocytes and ovarian tissue.

View larger version (15K): [in a new window]   Fig. 6. Properties of intercellular channels formed from Cx43.4 are similar to Cx45. N2A cells were transiently transfected with Xenopus Cx43.4 and analyzed by dual whole-cell voltage clamping. (A) Time-dependent inactivation of junctional currents in response to transjunctional potentials; 5 seconds long, 20 mV steps between -100 and +100 mV are shown. (B) Normalized steady-state conductance plotted against transjunctional voltage closely matches a double Boltzman distribution (N=7; V0=20 mV; Gmin/Gmax=0.05). (C) Single channel recordings display 35 pS transitions.

View larger version (38K): [in a new window]   Fig. 7. Spatial localization of Cx43.4 transcript in whole-mount neurula and tailbud stages. Whole-mount in situ hybridization revealed expression of Cx43.4 in dorsal structures extending from head to tail (A-G). By contrast, Cx30 was localized to ectoderm including the hatching gland and the endoderm (H-I). Embryos before clearing in benzyl benzoate/benzyl alcohol were photographed on a blue background. Cleared embryos are shown on yellow background. (A-C) The signal of Cx43.4 is strongly detected in the late neurula at stage 19. (A) Cx43.4 is in the neural folds and the eye vesicles, but not in the groove between the two neural folds (see white arrow). (B) The embryo from A was cleared to demonstrate localization of Cx43.4 in the posterior, already fused neural tube, as well in the anterior open tube, the brain and eyes. (C) A side view of the cleared embryo, anterior to the left, shows that Cx43.4 is restricted to dorsal neuroectoderm: head, spinal cord and tail. (D-F) Intensified accumulation of Cx43.4 in anterior structures of the tail bud embryo at stage 25. (D) One dark band of Cx43.4 expression in the anterior fused neural tube is marked with a white arrow. (E) A side view of two embryos (anterior to the left), demonstrate head, spinal cord and tail expression of Cx43.4 (F) A side view of a cleared embryo shows expansion of Cx43.4 expression in the head and brain structures as well in a branchial arch. Clear, but relatively lower expression is seen in the spinal cord and tail. (G) A side view of stage 33 tadpoles (anterior to the left) shows strong Cx43.4 expression in head and tail structures. As indicated earlier, expression is seen also in the spinal cord. (H) Whole mount staining shows Cx30 expression in the hatching glad and the anus. This superficial staining seems similar to the Cx43.4 staining seen in D. However, the side view of the cleared embryo in I (anterior to the left) shows that hatching glad staining is ectodermal staining and is different from the neural staining as seen for Cx43.4 (compare I to F and see the nueral staining in Fig. 8B). Another significant difference between the staining patterns of the two connexins is that, unlike Cx43.4, most of Cx30 expression is in the embryonic endoderm.

View larger version (126K): [in a new window]   Fig. 8. Spatial localization of Cx43.4 transcripts in sectioned wholemount tailbud embryos. (A) Transverse section through head reveals Cx43.4 in brain, eyes and head mesenchyme. (B) Similar section stained for N-CAM to confirm neural structures. (C,D) Posterior transverse sections show Cx43.4 and N-CAM in spinal cord. Bar (A), 200 µm.