Figure 5.
Figure 5. Sip1 knockdown leads to maintenance of Ecad protein in migratory NC cells. (A) Multiplex NanoString analysis of transcripts from neural tubes dissected from HH9 embryos, comparing the Sip1MO electroporated side to the control side of the same embryo. The results show that Ecad mRNA levels are up-regulated while Ncad and Cad7 are reduced (n = 3). The data shown here are representative of three separate experiments with the changes in transcript data pooled. (B) Quantitative RT-PCR analysis of neural tubes plus migratory crest cells dissected from HH9 embryos that were electroporated on the left side with ContMO (C) and on the right side with Sip1MO (S) at HH4. Ecad gene expression was compared with GAPDH expression. Error bars are standard deviation between samples and P < 0.036 (asterisk indicates statistical significance; n = 4). The data shown here are one representative experiment with expression data from four embryos, and this was repeated four times. (C) Western blot analysis of Ecad protein expression after the injection of 1 mM ContMO and 1 mM Sip1MO showing that Ecad protein expression is increased after Sip1MO electroporation. The data shown here are representative of one experiment out of three repeats. In this experiment, protein was pooled from 8–10 chicken whole embryos. β-Tubulin was used as a loading control. (D) In situ hybridization using a probe for Ecad shows that it is ectopically expressed in the neural tube after Sip1 knockdown (asterisk). Embryo injected on right side as indicated by asterisk. (E and F) Whole-mount immunohistochemistry for Ecad in midbrain sections at 40× in HH9 (E) and HH15 (F) embryos demonstrates increased sequestration of Ecad protein on the cell membranes on the Sip1MO side compared with the control side (white arrowheads). (G and H) To visualize the change in Ecad protein expression in vitro, neural tubes were dissected from HH8 embryos electroporated with ContMO on the left side and Sip1MO on the right side at HH4. Explants were cultured for 12–24 h and examined for expression of Ecad and HNK1. (G) ContMO-injected migratory neural crest cells express HNK1 (green) but lack Ecad expression (g′, white arrows), whereas cells lacking Sip1 (H) emigrate from the neural tube explants (marked by white dashed line) but fail to detach from each other or migrate far from the explant (n = 6/8). Additionally, the Sip1MO-injected cells co-express HNK1 (green) and Ecad protein (h′, white asterisk), suggesting that delamination from the neural tube explant does not require Ecad down-regulation, whereas subsequent detachment and migration does. (I and J) Whole-mount immunohistochemistry using an antibody for Pax7 in embryos electroporated with (I) ContMO on the left and Sip1 on the right (less migration on right) and (J) Sip1MO + EcadΔβcatenin on the right (recovered migration). Red dashed lines indicate midline and level of section. (i′) A section through the embryo in panel I demonstrating clumped Pax7-positive cells on the Sip1MO-injected side. (i′′) An overlay of Pax7 and FITC from ContMO and Sip1MO. (j′) A section of the embryo from J demonstrating recovered Pax7-positive cells on the Sip1MO + EcadΔβ-catenin–injected side. (j′′) An overlay of Pax7 and FITC Sip1MO. Bars: (D–J) 100 µm; (g′–h′′) 30 µm; (i′–j′′) 50 µm. The whole-mount and section data shown here are from a single representative experiment out of at least three repeats.

Sip1 knockdown leads to maintenance of Ecad protein in migratory NC cells. (A) Multiplex NanoString analysis of transcripts from neural tubes dissected from HH9 embryos, comparing the Sip1MO electroporated side to the control side of the same embryo. The results show that Ecad mRNA levels are up-regulated while Ncad and Cad7 are reduced (n = 3). The data shown here are representative of three separate experiments with the changes in transcript data pooled. (B) Quantitative RT-PCR analysis of neural tubes plus migratory crest cells dissected from HH9 embryos that were electroporated on the left side with ContMO (C) and on the right side with Sip1MO (S) at HH4. Ecad gene expression was compared with GAPDH expression. Error bars are standard deviation between samples and P < 0.036 (asterisk indicates statistical significance; n = 4). The data shown here are one representative experiment with expression data from four embryos, and this was repeated four times. (C) Western blot analysis of Ecad protein expression after the injection of 1 mM ContMO and 1 mM Sip1MO showing that Ecad protein expression is increased after Sip1MO electroporation. The data shown here are representative of one experiment out of three repeats. In this experiment, protein was pooled from 8–10 chicken whole embryos. β-Tubulin was used as a loading control. (D) In situ hybridization using a probe for Ecad shows that it is ectopically expressed in the neural tube after Sip1 knockdown (asterisk). Embryo injected on right side as indicated by asterisk. (E and F) Whole-mount immunohistochemistry for Ecad in midbrain sections at 40× in HH9 (E) and HH15 (F) embryos demonstrates increased sequestration of Ecad protein on the cell membranes on the Sip1MO side compared with the control side (white arrowheads). (G and H) To visualize the change in Ecad protein expression in vitro, neural tubes were dissected from HH8 embryos electroporated with ContMO on the left side and Sip1MO on the right side at HH4. Explants were cultured for 12–24 h and examined for expression of Ecad and HNK1. (G) ContMO-injected migratory neural crest cells express HNK1 (green) but lack Ecad expression (g′, white arrows), whereas cells lacking Sip1 (H) emigrate from the neural tube explants (marked by white dashed line) but fail to detach from each other or migrate far from the explant (n = 6/8). Additionally, the Sip1MO-injected cells co-express HNK1 (green) and Ecad protein (h′, white asterisk), suggesting that delamination from the neural tube explant does not require Ecad down-regulation, whereas subsequent detachment and migration does. (I and J) Whole-mount immunohistochemistry using an antibody for Pax7 in embryos electroporated with (I) ContMO on the left and Sip1 on the right (less migration on right) and (J) Sip1MO + EcadΔβcatenin on the right (recovered migration). Red dashed lines indicate midline and level of section. (i′) A section through the embryo in panel I demonstrating clumped Pax7-positive cells on the Sip1MO-injected side. (i′′) An overlay of Pax7 and FITC from ContMO and Sip1MO. (j′) A section of the embryo from J demonstrating recovered Pax7-positive cells on the Sip1MO + EcadΔβ-catenin–injected side. (j′′) An overlay of Pax7 and FITC Sip1MO. Bars: (D–J) 100 µm; (g′–h′′) 30 µm; (i′–j′′) 50 µm. The whole-mount and section data shown here are from a single representative experiment out of at least three repeats.

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