(A-B) Whole-mount in situ hybridization showing expression of e2f5 at 10-somite stage (10s) and 24 hpf. OP, olfactory placode; OV, otic vesicle; PD, pronephric duct. (C) Genomic structure and sequences of wild- type (wt) and three e2f5 mutant alleles. The underlined sequence in wt indicates PAM sequence of sgRNA target. (D) Phenotypes of male and female wild-type and e2f5 heterozygotes. Only fish exhibiting the male phenotype were present among homozygous mutants. (E) Bar graph showing the percentage of fertilization rates of wild-type, e2f5 homozygote and e2f5 homozygotes carrying Tg(β-actin:gfp-e2f5) transgene as indicated. The numbers of adult males investigated are listed at the bottom. (F) Diagram of the constructs for making gfp-e2f5 transgene. (G-H) H&E staining results showing testes of wild-type (G) and e2f5 homozygous mutants (H). Arrows indicate primary spermatocytes. Asterisks point to mature spermatozoa which were substantially reduced in the mutants. (I-J) Confocal images showing the staining of sperm flagella in wild-type (I) and e2f5 mutant testis (J) visualized with acetylated alpha-tubulin (ac-Tu) antibody. Nuclei and actin filaments were counterstained with DAPI and phalloidin, respectively. Scale bars: 1cm in panel D, 50 μm in panel G, H and 25 μm in panel I, J.

(A-H) Confocal images of primary spermatocytes at different prophase stages of meiosis I as indicated by anti-Sycp3 antibody staining (red). Arrowheads indicate spermatozoa nuclei from wild-type testis stained with DAPI in blue (A-E). (I) Bar graph showing the statistical results of the percentage of cells in different meiotic stages. The numbers of spermatocytes investigated are shown on top of each bar. (J-K) Staining of γH2AX (red) in the testes of wild-type and e2f5 mutant. Arrows point to the primary spermatocytes at leptotene and zygotene stages. (L-M) Confocal images showing apoptotic cells stained by TUNEL assay in red. (N) Bar graph with individual data points showing the number of TUNEL positive cells in wild-type and e2f5 mutant testes. (O) Phenotypes of e2f5;tp53 double mutants. (P) Bar graph showing the percentage of abnormal embryos produced from e2f5;tp53 double mutant females crossed with wild-type males. n = 441 from two double mutant females. (Q-T) Histological analysis of ovaries from wild-type and e2f5;tp53 double mutants. Inserted images are magnified views. Arrow in Q indicates peripheral localization of nucleoli stained by DAPI in stage II oocytes of wild-type ovary. The asterisk in S indicates cortical alveoli at later stage oocytes, which were visible under the confocal microscope due to autofluorescence. In panels J-M, Q-T, nuclei were counterstained with DAPI in blue. Scale bars: 10 μm in panel A-H, J-M and 50 μm in panel Q-T.

(A) Heat map of RNA-seq transcriptome analysis from two sets of wild-type and mutant testes. (B) Expression heat map of genes involved in homologous recombination in wild-type and e2f5 mutants. The full list of genes analyzed is listed in S2A Fig. (C) qRT-PCR results showing the relative expression level of genes involved in homologous recombination in wild-type and e2f5 mutant testes. The expression of each gene in wild-type testes was set as 100%. The full list of genes analyzed is listed in S2B Fig. (D) Diagram of the construct used for generating Tg(β-actin:dmc1) transgenic fish. (E) Dot plot showing the fertilization rate of e2f5 mutants carrying the Tg(β-actin:dmc1) transgene. (F-H) H&E staining results showing testes from wild-type, e2f5 mutant and e2f5 mutant carrying Tg(β-actin:dmc1) transgene. Asterisks indicate mature spermatozoa. Arrows indicate spermatocytes. (I-K) Confocal images showing the staining of sperm flagella visualized with an anti-acetylated tubulin antibody in green in wild-type and mutant testes as indicated. Nuclei and actin filaments were counterstained with DAPI (blue) and phalloidin (red) respectively. (L) CHIP analysis of GFP-E2f5 binding to the promoter of dmc1. (M) Diagram showing the position and sequence of potential E2f5 binding site near the transcription start site (TSS) of the dmc1 gene. (N) EMSA analysis of the interaction between E2f5 protein and oligonucleotide probes corresponding to the wild-type and mutant E2F binding sites as indicated in panel M. “++” indicates that the amount of probes was doubled than those in other parallel experiments (+). Scale bars: 100 μm in panel F-H and 25 μm in panel I-K.

(A-H) Confocal images showing cilia in the PST region of the pronephros of wild-type and mutant larvae at different stages as indicated. Cilia were visualized with anti-glycylated tubulin antibody in green. Nuclei were labeled with DAPI in blue. Red arrowheads indicate multicilia bundles and yellow arrowheads indicate single cilia. (I-J) Still images from S1 and S2 Movies showing cilia in the PST of 5dpf wild-type (I) and e2f4;e2f5 (J) double mutants. Bottom images show kymographs of cilia movement. (K) Beating frequency of cilia in the pronephric tubules of wild-type and e2f4; e2f5 double mutants. (L) Dot plot showing the width of pronephric tubule lumen as indicated by vertical lines in panel I and J. (M-P) Confocal images showing cilia and basal bodies labeled with anti-acetylated tubulin (ac-Tu, green) and anti-γ tubulin (γ-Tu, red) antibodies in the PST of 48 hpf wild-type and mutant larvae as indicated. Staining of γ-tubulin is also indicated in the bottom to show the multiple basal bodies of MCCs (red arrowheads) and single ciliary basal bodies (yellow arrowheads). (Q) The number of cells bearing motile cilia per arbitrary unit (a.u.) in the PST region of wild-type and mutant larvae as indicated. Scale bars: 25 μm in panels A-H, 10 μm in panels I, J and 5μm in panels M-P.

(A-E) Double fluorescence in situ hybridization results showing the expression of trpm7 and different motile cilia related genes as indicated. (F-J) Line profile plots showing the pixel intensities of green and red channels along the dotted line in panels A-E. (K) Confocal image showing cilia in the PST region of a 24 hpf wild-type larva. Cilia were visualized with anti-acetylated tubulin antibody in green and nuclei were counterstained with DAPI in blue. (L) Box plot graph showing relative length of 198 individual cilia from 33 embryos. Each dot represents a single cilium. Scale bars: 10 μm.

(A-P) Whole-mount in situ hybridization results showing the expression of e2f5 and other marker genes in the pronephric duct of 24 hpf control and mutant larvae as indicated. The numbers in the bottom right-hand corners indicate the numbers of embryos with similar staining results (left) and total numbers of embryos analyzed (right). (Q-V) Fluorescence in situ hybridization results showing the expression of rfx2 (red) and trpm7 (green) in the pronephric tubule of 36 hpf wild-type control (Q-S) and e2f5 mutant larvae (T-V). (W-X) Line profile plots showing the pixel intensities of green and red channels along the dotted line in panels S and V. (Y) Immunofluorescence results showing the staining of anti-acetylated tubulin (ac-Tu) and α6F antibodies on cross-sections through the pronephric tubules of 5dpf control and e2f5 mutant larvae. (Z) Model illustrating dual roles of E2f5 during multiciliogenesis. In the absence of E2f5 (maybe also E2f4), both MCC and principal progenitor cells developed a single motile cilium. Scale bars: 500 μm in panels A-P, 10 μm in panels Q-V and 5 μm in panel Y.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.