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FIGURE 5. Pronephros induction in animal cap assays. A, schematic diagram of in vitro pronephros induction assay showing animal caps are induced to
differentiate into pronephros tissues using atRA and activin. B and C, Hspa5MO2 (MO2) was injected into both blastomeres at the two-cell stage. Animal caps
were dissected at stage 9 and treated with activin and atRA for 3 h. Subsequently, the animal caps were transferred into normal animal cap culture mediumand
cultured until stage 15 (St15) (B) or stage 32 (St32) (C). Expression analysis was performed by RT-PCR using total RNA extracted from the animal caps. AC, control
animal cap; WE, sibling whole embryos. D and E, knockdown of Hspa5 suppresses lhx1 expression. Either 30 ng of Hspa5MO1 (MO1) (D) or Hspa5MO2 (MO2) (E)
was co-injected with 100 pg of LacZ mRNA into one ventral animal blastomere of four-cell stage embryos. Injected embryos were collected at stage 15. The
injected side was identified by red X-Gal staining, and the expression of lhx1 was examined by whole mount in situ hybridization. |
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FIGURE 3. Knockdown of Hspa5 inhibits pronephros formation. A–C, phenotype of Xenopus tadpoles injected with Hspa5MO2 into one ventral-vegetal
blastomere at the eight-cell stage. Either 15 or 30 ng of Hspa5MO2 was co-injected with LacZ mRNA into one of the ventral-vegetal blastomeres of eight-cell
stage embryos. A, anterior-dorsal view of mildly affected embryos at stage 16. B, lateral view of mildly affected embryos at stage 32. LacZ staining indicates the
injected side; the uninjected side was used as a negative control. C, phenotype categorization of embryos injected with 15 or 30 ng Hspa5MO2. Over 90% of
the embryos showed a normal or mild repression of anterior axis development after the injection. The severe phenotype refers to failure of gastrulation. D–F ,
depletion of Hspa5 inhibits expression of pan-pronephros marker genes such as pax2, lhx1, and atp1b1. Eight-cell stage embryos were injected with LacZmRNA
(100 pg) into one of the vegetal-ventral blastomeres as a lineage tracer along with Hspa5MO2 (30 ng/embryo). The injected embryos were collected at stage
32, and the expression of pronephric marker genes as indicated was examined by whole mount in situ hybridization. D, E, and F, the uninjected side. D , E , and
F , expression of pax2, lhx1, and atp1b1 on the MO-injected side of the same embryos, respectively. D , E , and F , transverse sections of embryos in D, E, and F
accordingly. G–H , expression of smp30 (tubule) and nephrin (glomus) at the Hspa5MO2-injected side was inhibited. G and H, the uninjected side. G and H ,
Hspa5MO2-injected side. G and H , transverse sections of embryos shown in G and H, respectively. I–L , expression of pax2, lhx1, smp30, and nephrin in the
tadpoles injected with 30 ng of standard control MO/embryo with 100 pg of LacZ mRNA into one vegetal-ventral blastomere at the eight-cell stage. I–L,
uninjected side. I -–L , standard control MO-injected side (CMO-lacZ). |
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FIGURE 8. Lhx1 expression partially rescues pronephros inhibition induced by Hspa5MO. A–H , expression of pronephric markers including lhx1, pax2,
nephrin, and smp30 in embryos injected with either Hspa5MO1 (MO1) (30 ng) or a mixture of Hspa5MO1 (30 ng) and lhx1 (100 pg) were examined by whole
mount in situ hybridization. Single ventral-vegetal blastomeres of eight-cell stage embryos were targeted, and LacZ mRNA was used as a lineage tracer. A, C, E,
and G, Hspa5MO-injected side, A , C , E , and G , non-injected (Non-inj) side of the same embryo. B, D, F, and H, Hspa5MO1- and lhx1-injected side. B , D , F , and
H , non-injected side. I, quantification of the rescue experiments of different pronephros markers. The phenotype assessments were performed blinded to the
treatment. The numbers on the tops of the columns indicate the total number of injected embryos. |
