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Fascin is a conserved protein that has been shown to modulate the cytoskeleton. Its role in early development remains unclear. After fertilization, embryos undergo rapid cell divisions, requiring the precise regulation of cytoskeleton to segregate chromosomes. Results indicate that Fascin is in the cell cortex, enriched in the perinuclear region of non-dividing blastomeres and on the mitotic spindle of dividing blastomeres of the early embryo. Loss-of-function of Fascin leads to a significant developmental delay or arrest, indicating that Fascin is important for proper early embryonic development.
Figure 1. Knockdown of Fascin leads to delayed or arrested developmental progression .
(A) Egg, zygote, 2-cell, 4-cell, 8-cell, 16-32-cell embryos were immunolabeled with Fascin antibody (green) to examine levels of Fascin protein through development. Tukey Kramer statistical test (Pollard et al., 2019) was used to analyze Fascin protein levels among various developmental stages. *denotes p<0.05. NS=not significant. (B) Control or Fascin MASO-injected (2 mM) embryos were immunolabeled with Fascin antibody (green) and counterstained with DAPI to label the DNA (blue). Fascin protein is localized perinuclearly in non-dividing blastomeres, enriched on the mitotic spindle in dividing blastomeres (arrows), and at the cell cortex. Fascin protein in Fascin MASO-injected embryos is significantly decreased compared to the control MASO-injected embryos. Scale bar = 50 µm; 3 biological replicates. (C) The level of Fascin protein in control and Fascin MASO-injected (2 mM) in individual blastomere of the 16-32 cell stage embryos was quantified with ImageJ, by using the average fluorescence per blastomere. Fascin protein is significantly decreased in embryos injected with the Fascin MASO compared to the control, demonstrating the efficacy of the Fascin knockdown. Circles represent individual measurements corresponding to each biological replicate. N=total number of blastomeres measured; 3 biological replicates; *denotes p<0.005 using a Student’s
t
-test comparing all measurements from 3 biological replicates. (D) The specificity of the Fascin antibody is tested with pre-adsorption assay. Fascin coding sequence is cloned into hemagglutinin (HA) and histidine (His)-tagged expression vector and transformed into C41 cells (Lucigen, Middleton, WI). Western blot indicates Fascin-HA-His tagged protein (37.98 kDa) is recognized with an anti-HA antibody. Bacterial lysates expressing Fascin-HA-His were used to conduct pre-adsorption tests. Embryos were immunolabeled with Fascin antibodies pre-adsorbed with lysate from bacteria expressing HA-tagged Fascin or human Rab35-GST. Embryos immunolabeled with pre-adsorbed Fascin antibodies have decreased Fascin (shown in green) compared to embryos immunolabeled with non-pre-adsorbed Fascin antibody. (E) Zygotes were injected with control or Fascin MASO at 0.5mM, 1mM, and 2mM. The percentage of embryos at a specific stage was recorded hourly from 2 to 6 hours post fertilization (hpf). Loss-of-function of Fascin results in developmental delay or arrest in early cleavage stage embryos in a dose-dependent manner. Error bars depict standard error of the mean (SEM). 3 biological replicates. *denotes p< 0.01 using Cochran-Mantel-Haenszel test (Mantel & Haenszel, 1959). N=number of embryos counted per timepoint.
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