|
|
| |
Fig. 3 – Defective blood flow through the vasculature caused
by loss of xkindlin2. (A–B) Images of tadpoles at indicated
time intervals following injection of fluorescent dextran into
the beating hearts of an uninjected (UN) tadpole and a K2MO
injected tadpole. The top row are brightfield images and
three bottom rows are fluorescence micrographs showing
progressive distribution of the dextran; (k) kidneys. (C–F)
RNA in situ hybridizations using hba1 as a blood marker.
(C–D) Early specification of blood islands appeared normal in
both ConMO and K2MO morphants. (E–F) At stg 40, discrete
blood islands were visible in ConMO morphants; K2MO
morphants showed prominent staining of hba1 in a
branched pattern. |
|
Display additional annotations [+]
|
| |
Fig. 2 – Blood cell aggregation, ventral edema and pale hearts caused by loss of xkindlin2. (A) Sequence of K2MO used to target
both alleles of xkindlin2. (B) Efficacy of K2MO mediated knockdown of endogenous xkindlin2; lysates from three
developmental stages were immunoprecipitated using anti-human kindlin2 Ab (3A3.5) to enrich for xkindlin2. Western blot
was performed using anti-mouse kindlin2 Ab (Y-15). K2MO (injected post-fertilization) failed to knockdown xkindlin2 at
stage 11 but reduced xkindlin2 expression evident by stage 26. Recovery of xkindlin2 expression in these embryos is noted
by stage 40. (C) Quantification of xkindlin2 expression in the presence of ConMO and K2MO at stage 26. N = 3, error
bars = SEM. (D–E) Phenotypes of ConMO and K2MO injected tadpoles; blood smears (red asterisks) and ventral edema (ve)
evident in the K2MO morphant. (F–G) RNA in situ hybridization using hba1 as a blood marker confirmed presence of blood
smears in K2MO embryos. (H–I) Micrographs of tadpole heads viewed from the ventral side to visualize the heart (h); red
blood cells are visible in hearts of ConMO but not in K2MO morphant hearts. (J–K) RNA in situ hybridizations using nkx2–5 as a
marker for heart differentiation. (L) Injection of non-targetable EGFP-tagged human kindlin2 RNA rescued the most severe
defects (i.e., embryos with both ventral edemas and pale hearts) caused by injection of K2MO. (N = 4, n = 163–180 embryos,
error bars = SEM). |
