E2f5 is required for the formation of MCCs in the pronephric duct. A: Schematic showing CRISPR/Cas9-mediated genome editing at the zebrafish e2f5 locus using two guide RNAs targeting exon 1 and exon 5. After non-homologous end joining (NHEJ), F1 carriers contained a 37-bp deletion in exon 1, and a subsequent 10-bp deletion in exon 5. This translates to a truncated protein of 117-amino acid. B: Kidney tubule of a 48 hpf wild-type embryo populated with clusters of MCCs (arrows). C: Kidney tubule of an e2f5 mutant embryo completely devoid of MCCs. Cilia from monociliated cells, which are not affected by the loss of e2f5, are indicated (arrows). Acetylated-tubulin (Ace-tub; green), DAPI (blue). Scale bars= 10 µm. D: MCCs around the lateral rim of a nasal placode of a wild-type embryo at 72 hpf (arrow). E: Reduction in the number of nasal placode MCCs in e2f5 homozygous mutant (arrow). Acetylated-tubulin (Ace-tub; green); γ-tubulin (γ-tub; red). L: lateral; M: medial. Scale bars = 10 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

E2f4 is dispensable for MCC formation. A: Schematic showing CRISPR/Cas9-mediated genome editing at the zebrafish e2f4 locus using two guide RNAs targeting exon 1 and exon 6. After NHEJ, F1 carriers contained a 462-bp deletion between exon 1 and exon 6. This resulted in a 154-amino acid in-frame deletion. B: Kidney tubule of a 48 hpf wild-type embryo populated with clusters of MCCs (arrows). C: MCCs are unaffected in the kidney tubule of an e2f4 homozygous mutant (arrows). Acetylated-tubulin (Ace-tub; green), DAPI (blue). Scale bars: 10 µm. D: Kidney tubule of an e2f5 mutant embryo completely devoid of MCCs. E: Rescued MCCs in the kidney tubule of an e2f5 mutant by over-expression of E2f4 (arrows). Acetylated-tubulin (Ace-tub; green), DAPI (blue). Scale bars= 10 µm. F: MCCs around the lateral rim of a nasal placode (arrow) of a wild-type embryo at 72 hpf. G: Nasal placode MCCs are unaffected in an e2f4 mutant (arrow). Acetylated-tubulin (Ace-tub; green), γ-tubulin (γ-tub; red). L: lateral; M: medial. Scale bars = 10 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Both e2f4 and e2f5 contribute to MCC formation at the nasal placode. A: Thick layers of motile cilia bundles (labelled by anti-acetylated α-tubulin (Ace-tub)) were featured prominently on the lateral side of e2f4^+/- nasal placodes. MCCs can be identified by patches of densely packed basal bodies (labelled by anti-gamma-tubulin (γ-tub)), a subset of which is featured in the single channel inset (white dotted rectangle, image scale unchanged). B: e2f4^-/-; e2f5^-/- double homozygous mutants show a complete lack of MCCs. C: MCCs are essentially absent in the nasal placodes of e2f4^+/-; e2f5^-/- embryos. D: The nasal placode MCC layers are present but much thinner in e2f4^+/-; e2f5^-/- mutants than that in wild-type or heterozygous siblings. All embryos were siblings from an inter-crosses of e2f4^+/-; e2f5^+/- adult mutants and were fixed for immunostaining at 72 hpf. L: lateral; M: medial. Scale bar = 10 µm.

Effects of combinatorial loss of E2f4 and E2f5 on the MCC transcriptional program in the pronephros and nasal placode. A-D: At 48 hpf, expression of mci (A, B) as well as foxj1b were completely absent in e2f4^+/-; e2f5^-/- pronephros (C, D). Regions populated with positively labelled cells are flanked by black arrowheads. E, F: At 72 hpf, foxj1a is exclusively expressed on the lateral side of the nasal placodes (E), but was absent in e2f4^+/-; e2f5^-/- (F) embryos. G, H: At 72 hpf, foxj1b is expressed more widely than foxj1a, found both on the lateral side and within the placode. While the signals on the lateral side (where MCCs predominantly reside, black arrows) were absent in e2f4^+/-; e2f5^-/- (black arrows), staining of non-MCC cells remained unchanged (white arrowheads). I-L: foxj1a expression was absent at 72 hpf in gmnc embryos (I, J), where similar loss of foxj1b on the lateral side was also observed (K, L). Scale bars = 20 µm.

Whole mount in situ hybridization staining of e2f4 at 24 and 36 hpf of embryonic development. A: e2f4 expression in the cephalic region at 24 hpf. B: e2f4 expression in the trunk at 24 hpf. C: e2f4 expression in the cephalic region at 36 hpf. D: e2f4 expression in the trunk at 36 hpf. Scale bars = 200µm.