Figure 4.
Figure 4. CA disrupts ciliogenesis and mitotic spindle morphology. (A) Immunostaining of E15.5 kidney sections from Hoxb7-Plk4, Six2-Plk4, and control embryos with antibodies against γ-tubulin (centrosomes), acetylated α-tubulin (cilia; Ac. tubulin), CK8 (UB epithelia), Six2 (MM), and DAPI (DNA). Normal kidney cells contain one centrosome and cilium, whereas progenitor cells with excess centrosomes are either aciliated or superciliated. (B) Quantification of ciliary assembly defects in UB cells of Hoxb7-Plk4 mice or MM cells of Six2-Plk4 mice. The percentage of cells with zero, one, or more than one cilium was determined from cells that contained normal centrosome number compared with cells with CA. n = 311 cells (E15.5 WT control; normal centrosomes), 263 cells (E15.5 Hoxb7-Plk4; amplified centrosomes), 253 cells (E15.5 WT control; normal centrosomes), and 238 cells (E15.5 Six2-Plk4; amplified centrosomes) from five mice of each genotype per developmental stage. Statistical significance was determined by a two-tailed unpaired t test (*, P < 0.05). (C) Representative images from E15.5 kidney sections of control, Hoxb7-Plk4, and Six2-Plk4 embryos immunostained with antibodies against RET, phospho-ERK, WNT11, LEF1, E-cadherin, and DAPI. Bars (insets), 5 µm. (D) Graphs showing relative levels of RET, pERK, and WNT11 in ureteric buds of E15.5 WT, Hoxb7-Plk4, or Six2-Plk4 mice. The fluorescence intensity was quantified by outlining each UB structure and the signal intensity measured and represented per unit area. For MM differentiation, the number of LEF1-positive cells per renal vesicle (adjacent to E-cadherin–positive UB structures) was scored. n = 4 for WT, Hoxb7-Plk4, and Six2-Plk4 mice. A two-tailed unpaired t test was used for statistical analysis (*, P < 0.05). (E) Representative images of E15.5 kidney sections from Six2-Plk4 and control mice stained for centrosomes (γ-tubulin), mitotic spindle microtubules (α-tubulin), mitotic marker (pHH3), and DNA (DAPI). In control kidneys, two centrosomes organize bipolar spindles that will eventually segregate the DNA into two daughter cells. When extra centrosomes are present, multipolar configurations are frequent (right). When centrosome clustering occurs (middle), the morphology of mitotic spindles appears bipolar (termed pseudobipolar). Bars (insets), 1 µm. (F) Quantification of the percentage of cells with bipolar and multipolar spindle configurations over time. In control animals that contain the normal complement of centrosomes, cells only form bipolar spindles in mitosis. To determine spindle morphologies in Hoxb7-Plk4 and Six2-Plk4 animals, quantification was done only in cells that contained excess centrosomes, as cells with normal centrosome number only form normal bipolar spindles (not depicted). In cells with CA, multipolar spindles are prevalent at early stages of development in both Hoxb7-Plk4 and Six2-Plk4 animals. However, cells with supernumerary centrosomes display pseudobipolar spindle morphologies at later development stages. n = 5 mice of each genotype from each developmental time point. A two-tailed unpaired t test was used to determine statistical significance (*, P < 0.05).

CA disrupts ciliogenesis and mitotic spindle morphology. (A) Immunostaining of E15.5 kidney sections from Hoxb7-Plk4, Six2-Plk4, and control embryos with antibodies against γ-tubulin (centrosomes), acetylated α-tubulin (cilia; Ac. tubulin), CK8 (UB epithelia), Six2 (MM), and DAPI (DNA). Normal kidney cells contain one centrosome and cilium, whereas progenitor cells with excess centrosomes are either aciliated or superciliated. (B) Quantification of ciliary assembly defects in UB cells of Hoxb7-Plk4 mice or MM cells of Six2-Plk4 mice. The percentage of cells with zero, one, or more than one cilium was determined from cells that contained normal centrosome number compared with cells with CA. n = 311 cells (E15.5 WT control; normal centrosomes), 263 cells (E15.5 Hoxb7-Plk4; amplified centrosomes), 253 cells (E15.5 WT control; normal centrosomes), and 238 cells (E15.5 Six2-Plk4; amplified centrosomes) from five mice of each genotype per developmental stage. Statistical significance was determined by a two-tailed unpaired t test (*, P < 0.05). (C) Representative images from E15.5 kidney sections of control, Hoxb7-Plk4, and Six2-Plk4 embryos immunostained with antibodies against RET, phospho-ERK, WNT11, LEF1, E-cadherin, and DAPI. Bars (insets), 5 µm. (D) Graphs showing relative levels of RET, pERK, and WNT11 in ureteric buds of E15.5 WT, Hoxb7-Plk4, or Six2-Plk4 mice. The fluorescence intensity was quantified by outlining each UB structure and the signal intensity measured and represented per unit area. For MM differentiation, the number of LEF1-positive cells per renal vesicle (adjacent to E-cadherin–positive UB structures) was scored. n = 4 for WT, Hoxb7-Plk4, and Six2-Plk4 mice. A two-tailed unpaired t test was used for statistical analysis (*, P < 0.05). (E) Representative images of E15.5 kidney sections from Six2-Plk4 and control mice stained for centrosomes (γ-tubulin), mitotic spindle microtubules (α-tubulin), mitotic marker (pHH3), and DNA (DAPI). In control kidneys, two centrosomes organize bipolar spindles that will eventually segregate the DNA into two daughter cells. When extra centrosomes are present, multipolar configurations are frequent (right). When centrosome clustering occurs (middle), the morphology of mitotic spindles appears bipolar (termed pseudobipolar). Bars (insets), 1 µm. (F) Quantification of the percentage of cells with bipolar and multipolar spindle configurations over time. In control animals that contain the normal complement of centrosomes, cells only form bipolar spindles in mitosis. To determine spindle morphologies in Hoxb7-Plk4 and Six2-Plk4 animals, quantification was done only in cells that contained excess centrosomes, as cells with normal centrosome number only form normal bipolar spindles (not depicted). In cells with CA, multipolar spindles are prevalent at early stages of development in both Hoxb7-Plk4 and Six2-Plk4 animals. However, cells with supernumerary centrosomes display pseudobipolar spindle morphologies at later development stages. n = 5 mice of each genotype from each developmental time point. A two-tailed unpaired t test was used to determine statistical significance (*, P < 0.05).

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