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. 2014 Mar 27;25(9):1991–2002. doi: 10.1681/ASN.2013090976

Figure 5.

Figure 5.

Phenotype of anillin knockdown in zebrafish. (A) ANLN knockdown leads to an edema phenotype in zebrafish (pericardial effusion, arrow; yolk sac, arrowhead). Fertilized eggs are injected with a control or Anillin-specific splice-donor morpholino and phenotypes are scored at 120 hours post fertilization (hpf). Edema development is graded as follows: I, no phenotype; II, mild phenotype; III, severe; and IV, very severe phenotype. (B) Anillin-knockdown leads to loss of injected high molecular mass dextran. Zebrafish larvae 48 hpf postinjection of no morpholino (WT), control morpholino (Ctrl), or Anillin morpholino are anesthetized and injected with a FITC-labeled 70-kD dextran. The amount of systemic fluorescence is assessed postinjection by measurement of fluorescence intensity in the retinal blood vessel plexus at baseline and 24 hours later in individual fish. Loss of systemic fluorescence indicates systemic loss of high molecular mass proteins from the circulation, especially in fish with a severe edema phenotype (PIII/PIV). (C) Anillin-knockdown leads to loss of systemic fluorescence in Tg (l-fabp:DBP-EGFP) zebrafish. Tg (l-fabp:DBP-EGFP) zebrafish develop from 96 hpf until 144 hpf, increasing systemic fluorescence by increasing amounts of circulating EGFP-labeled vitamin D binding protein (molecular mass of approximately 64 kD). Anillin knockdown causes a significant reduction in systemic fluorescence in mild (phenotype I/II) and severely affected (phenotype III/IV) knockdown fish, indicating again systemic loss of high molecular mass proteins. A dot blot assay of DBP-EGFP expression in fish water from anillin-knockdown zebrafish shows strongly detectable fluorescence compared with both control and wild-type fish. (D) TEM of the GFB. In the control morpholino-injected embryo, podocyte foot processes and intervening slit diaphragm architecture are preserved (boxed area in Da and white arrow in Da’). In the anillin-knockdown fish, there is effacement and fusion of the podocyte foot process with disruption of the slit diaphragm (boxed area in Db and white arrow head in Db’). Bar, 500 nm in D.