|
| Gene |
Clone |
Species |
Stages |
Anatomy |
|
dag1
|
|
laevis
|
NF stage 23
|
otic placode
,
optic vesicle
,
epibranchial placode
,
pronephric kidney
,
anterior
|
|
dag1.L
|
|
laevis
|
NF stage 28
|
otic vesicle
,
pronephric duct
,
branchial arch
,
paraxial mesoderm
,
pronephric kidney
|
|
dag1.L
|
|
laevis
|
NF stage 31
|
otic vesicle
,
midbrain-hindbrain boundary
,
pronephric duct
,
branchial arch
,
paraxial mesoderm
,
[+]
|
|
dag1.L
|
|
laevis
|
NF stage 35 and 36
|
midbrain-hindbrain boundary
,
pronephric nephrostome
,
pronephric duct
,
branchial arch
,
pronephric kidney
|
|
| |
Fig. 1. Dystroglycan expression during Xenopus laevis pronephros development. Panels A–D show spatial expression of dystroglycan transcripts from stage 23 to 35, and schematic representation of the developing pronephros adapted from Vize et al. (1995). Whole-mount in situ hybridizations were performed using digoxigenin-labelled antisense αDg RNA probes. (A) Stage 23. Expression is confined to the developing pronephric tubule anlage. (B–D) Stage 28, 31 and 35. Dg transcripts are present in the nephrostomes, the pronephric tubules (red arrowhead) and in the pronephric duct (green arrowhead). Panels E–H show immunolocalization of Dg protein. Embryonic stages are indicated in the top right of each panel. (E) Stage 35. Dg is present in the pronephric tubules (red arrowhead), in the anterior and posterior segments of the pronephric duct (blue and green arrowheads). (F–H) Cryostat sections. (F) Stage 29/30. Dg protein is detected on the surface of cells into the pronephric bud (yellow arrowhead) and at the basal pole of cells surrounding the pronephric bud (white arrowhead). (G) Stage 36. The Dg is detected around the nephrostomes (yellow arrowhead) and the pronephric tubules (red arrowhead). (H) Stage 40. The Dg is present in the elongating tubules (white arrowhead), and duct (yellow arrowhead). |
|
|
| |
Fig. 2. Depletion of dystroglycan by antisense morpholino oligomers. (A) The positions of the antisense morpholino oligomers and ATG initiation codon are indicated. (B) Dg-Mo specificity in vivo; Western Blot analysis using anti-Dg antibody showing that the Dg-Mo interferes with in vivo translation in Xenopus embryos of Dg mRNA. Lane 1, uninjected embryos; Lanes 2–4, embryos were injected in both blastomeres at the two cell stage with 8 ng (lane 2), 16 ng (lane 3) and 18 ng (lane 4) of Dg-Mo1 + 2. At stage 28 dorsal tissues containing pronephric anlage were isolated and proteins were analysed by SDS-PAGE. α-Tubulin was used as a loading control. Significant reduction of Dg protein synthesis is observed between 8 ng and 18 ng of morpholino. (C) Frequency of the phenotypes obtained with the different concentrations of Mo analysed with the 3G8 and 4A6 antibodies. The number n indicates the total number of injected embryos. (D–G) Whole-mount immunodetection of Dg protein at stage 41 on embryos injected unilaterally at the 4-cell stage with Dg-Mo1 + 2 (16 ng, panel E and 18 ng, panel G in both left blastomeres). At the dose of 16 ng Dg-Mo1 + 2 a strong reduction of Dg immunoreactivity is observed in the injected side (E) compared to the control side (D). At 18 ng Dg-Mo1 + 2, Dg immunoreactivity is absent in the injected side (G) compared to the control (F). (H–K) The phenotype of pronephric tubules was rescued by coinjection with Dg full-length mRNA. (H, I) Pronephric tubule formation is inhibited in the Dg-Mo injected side of embryos (arrowhead in panel I) compared to the non-injected-side (arrowhead in panel H). (J–K) Pronephric tubule formation was significantly rescued in embryos injected with Dg-Mo1 + 2 (16 ng) plus Dg full-length (FL) mRNA (400 pg) (arrowhead in panel K) compared to the Dg-Mo1 + 2 injected side (arrow in panel I). |
