(A) Klf4 is expressed in the epiblast of the deep cell layer (DEL), yolk syncytial layer (YSL), and enveloping layer cells (EVL) of 60%, 70%, 80% and 90% epiboly embryos. Insets show respective XZ or YZ projections of confocal images from an 80% epiboly embryo along the axes shown in the main panel (d). Klf4 is expressed in the ectoderm and EVL of bud (tb), 5s and 10s embryos. (B) Images of p63 expression during different embryonic developmental stages are shown (a-d). Colocalization of Klf4 and p63 was detected in ventral ectoderm of embryos at various stages, including 90% epiboly, bud, 5s and 10s (e-h). Enlargements of Klf4 and p63 merged images (e-h) are shown from the areas indicated in lower magnification images (a-d). Percentage of colocalization of Klf4 and p63 in respective stage is indicated within each panel. Some non-colocalized cells showing only expression of Klf4 (arrowhead) or p63 (arrow) were observed in the ventral ectoderm of embryos at 5s and 10s. Insets show YZ projections of confocal images. (C) Images of wild-type embryos stained with anti-Klf4 antibody and dlc antisense RNA at 90% epiboly, bud and 5s stages are shown. Enlargements of merged images (d, h, l) show the areas indicated in corresponding lower magnification pictures (c, g, k). Insets show YZ projections from confocal images. Arrowheads indicate examples of ionocyte progenitors with colocalized Klf4 and dlc in the epidermal ionocyte domain. Arrows indicate ionocyte progenitors expressing only dlc. dlc⁺ ionocyte progenitor number at indicated embryonic stages is shown (m). Scale bar, 50 μm. Error bars indicate standard error.

(A) (a) Partial nucleotide sequence of wild-type (reference) klf4, showing sgRNA (green lettering) targeted to exon 4 is shown. The klf4^d5i1 mutant had a 1 bp insertion (red lettering) and 5 bp deletion (dashed line). Protospacer adjacent motif (PAM) sequence is shown in blue. (b) Predicted amino acid sequence of klf4 with the first zinc finger motif in wild type is compared to the misframed zinc finger domain and stop codon (red lettering) in klf4^d5i1 mutants. (B) Images of BrdU-labeled klf4^+/+, klf4^+/- or klf4^-/- embryos, followed by staining with dlc antisense RNA, and stained with anti-p63 and anti-BrdU antibodies at bud stage are shown. Both p63⁺ and p63⁺ BrdU⁺ cell number were enumerated in the circled area of klf4^+/+, klf4^+/- or klf4^-/- embryos. Enlarged images of klf4^+/+, klf4^+/- or klf4^-/-embryos stained with p63 and BrdU or dlc RNA probe and BrdU are shown. Examples of p63 and BrdU or dlc and BrdU colocalized (arrowhead) or non colocalzed (arrow) cells are shown. (C) Quantitative results from (B). Total p63⁺ or dlc^- p63⁺ cell numbers (open bars) with BrdU⁺ cell numbers (filled bar) of klf4^+/+, klf4^+/- or klf4^-/- embryos at bud stage are shown in (a) and (b). dlc⁺p63⁺ or dlc⁺p63^- cell numbers (open bars) with BrdU⁺ cell numbers (filled bars) of klf4^+/+, klf4^+/- or klf4^-/- embryos at bud stage are shown in (c) and (d). Statistical significance is indicated for comparisons of total cell numbers (open box) or BrdU⁺ cell numbers (filled box). Individual percentages of p63⁺BrdU⁺, dlc^- p63⁺ BrdU⁺, dlc⁺ p63⁺ BrdU⁺ or dlc⁺ p63^- BrdU⁺ cells of klf4^+/+, klf4^+/- or klf4^-/- embryos at bud stage are indicated within the bar. Embryos are shown in lateral view. Statistical significance was determined by Student’s t-test. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Error bars indicate standard error.

(A) The cell density of dlc⁺ ionocyte progenitors was reduced in embryos injected with both klf4 MO1 and MO2 (c) as compared to uninjected wild type (a) or control embryos injected with klf4 5mmMO2 (b) at bud stage. Cell densities of dlc⁺ ionocyte progenitors were quantified from the indicated area in wild-type embryos and embryos injected with klf4 5mmMO2 or both klf4 MO1 and MO2 (d). The density of dlc⁺ ionocyte progenitors was increased in klf4-overexpressing embryos (g) compared to LacZ-overexpressing (f) or uninjected wild-type (e) embryos. Cell density of dlc⁺ ionocyte progenitors was quantified in wild-type embryos and LacZ or klf4 mRNA-injected embryos (h). (B) The density of foxi3a⁺ ionocyte progenitors was reduced in embryos injected with both klf4 MO1 and MO2 (c), as compared to uninjected wild-type (a) or control embryos injected with klf4 5mmMO2 (b) at 5s stage. Cell densities of foxi3a⁺ ionocyte progenitors were quantified from the indicated area in wild-type embryos and embryos injected with klf4 5mmMO2 or both klf4 MO1 and MO2 (d). The density of foxi3a⁺ ionocyte progenitors was increased in klf4-overexpressing embryos (g), as compared to LacZ-overexpressing (f) or uninjected wild-type (e) embryos. Cell densities of foxi3a⁺ ionocyte progenitors were quantified in wild-type embryos and LacZ- or klf4-injected embryos (h). (C) The density of foxi3b⁺ ionocyte progenitors was reduced in embryos injected with both klf4 MO1 and MO2 (c) as compared to uninjected wild-type (a) or control embryos injected with klf4 5mmMO2 (b) at 5s stage. Cell density of foxi3b⁺ ionocyte progenitors was quantified from the indicated area in wild-type embryos and embryos injected with klf4 5mmMO2 or both klf4 MO1 and MO2 (d). The density of foxi3b⁺ ionocyte progenitors was increased in klf4-overexpressing embryos (g) compared to LacZ-overexpressing (f) or uninjected wild-type (e) embryos. Cell density of foxi3b⁺ ionocyte progenitors was quantified in wild-type embryos and LacZ or klf4 mRNA injected embryos (h). Embryos are shown in lateral view. Statistical significance was determined by Student’s t-test. *p < 0.05; ***p < 0.001. Scale bar, 100 μm. Error bars indicate standard error.

(A) The density of atp1a1a.1⁺-Na⁺-K⁺-ATPase-rich (NaR) cells was reduced in yolk extensions of embryos injected with both klf4 MO1 and MO2 (c) as compared to uninjected wild types (a) and control embryos injected with klf4 5mmMO2 (b). The density of atp1a1a.1⁺-NaR cells was increased in yolk extensions of embryos injected with klf4 (g) mRNA, as compared to embryos injected with the same amount of LacZ (f) mRNA, or uninjected wild type (e) embryos. Quantification of results from (a-c) and (e-g) are shown in (d) and (h), respectively. (B) The density of atp6v1aa⁺- H⁺-ATPase-rich (HR) cells was reduced in yolk extensions of embryos injected with both klf4 MO1 and MO2 (c) as compared to uninjected wild types (a) and control embryos injected with klf4 5mmMO2 (b). The density of atp6v1aa⁺-HR cells was increased in yolk extensions of embryos injected with klf4 (g) mRNA, as compared to embryos injected with the same amount of LacZ (f) mRNA, or uninjected wild-type (e) embryos. Quantification of results from (a-c) and (e-g) are shown in (d) and (h), respectively. Statistical significance was determined by Student’s t-test. ***p < 0.001. Scale bar, 300 μm. Error bars indicate standard error.

(A) BrdU and p63 labeling were performed on embryos injected with klf4 5mmMO2 (control) (a-e), combined klf4 MO1/klf4 MO2 (f), p53 MO (g), combined cdkn1a MO/ klf4 MO1/klf4 MO2 (h), combined p53 MO/klf4 MO1/klf4 MO2 (i), combined klf4-7mm mRNA/klf4 MO1/klf4 MO2 (j), or combined klf4ΔC-7mm mRNA/klf4 MO1/klf4 MO2 (k). Nuclei are counterstained with Hoechst 33342 (c). Both p63⁺ and p63⁺BrdU⁺ cell number were enumerated in the circled areas. Examples of p63 and BrdU colocalized cells (asterisk) are shown. The percentage of p63⁺BrdU⁺ cells in embryos after indicated treatments was quantified (l). (B) p53 expression in embryos injected with combined klf4 MO1/klf4 MO2, combined klf4-7mm mRNA/klf4 MO1/klf4 MO2, or combined klf4ΔC-7mm mRNA/klf4 MO1/klf4 MO2 was measured by RT-qPCR. (C) cdkn1a/p21 expression in embryos after indicated treatments was measured by RT-qPCR. Statistical significance was determined by Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar, 50 μm. Error bars indicate standard error.

(A) Four putative KLF-binding motifs were identified in the upstream region of the dlc promoter. The core binding motif (CACCC) is indicated by red text (a). Chromatin immunoprecipitation was performed using anti-Klf4 or anti-Myc antibodies, and qPCR of the isolated chromatin revealed significant enrichment of the KLF-binding motif located between -756 to -747 of the dlc promoter (b). (B) A diagram depicting the organization of dlc11k-mCherry, dlc3k-mCherry, and dlc3kM-mCherry constructs. KLF binding motif is shown in red text, and mutated bases are indicated by lowercase letters (a). Images of 5s embryos labeled with dlc antisense RNA probe (b), F1 transgenic embryos from Tg(dlc11k:mCherry) (c-e), Tg(dlc3k:mCherry) (f, g) and Tg(dlc3kM:mCherry) (h, i) are shown. Five somites are indicated (e). (C). Uninjected (un-inj) Tg(dlc3k:mCherry) (a) or Tg(dlc3kM:mCherry) (e), LacZ-injected Tg(dlc3k:mCherry) (b) or Tg(dlc3kM:mCherry) (f) and klf4-injected Tg(dlc3k:mCherry)(c) or Tg(dlc3kM:mCherry) (g) embryos were stained with anti-mCherry antibody at bud stage. Quantification of results from (a-c) is shown (d). Statistical significance was determined by Student’s t-test. ***p < 0.001. Error bars indicate standard error. CG, cranial ganglia; EI, epidermal ionocytes; S, somites; PSM, presomitic mesoderm.

Zebrafish Klf4 exerts two-level regulatory activity toward the development of ionocytes. In wild-type embryos, Klf4 represses p53 expression, preventing the induction of cdkn1a/p21, and thereby allowing proper p63⁺ epidermal stem cell proliferation (box 1). At a second level, Klf4 acts as a repressor, regulating Dlc-mediated lateral inhibition by binding to the dlc promoter to maintain proper dlc⁺ ionocyte progenitor population and patterning during the initiation of ionocyte progenitor differentiation (box 2). The output patterns are illustrated in 2D cell schematics, and representative images of dlc mRNA in situ hybridization from klf4 morphants, wild-type and klf4-overexpressing embryos at bud stage are shown in box 3. Under klf4 deficiency, p53 expression is no longer repressed and cdkn1a/p21 is activated, which results in reduced proliferation of p63⁺ epidermal stem cell and larger/fewer dlc⁺ progenitors after selection by lateral inhibition (box 3.1). When klf4 is overexpressed (box 3.3), more stem cells are selected as ionocyte progenitors and dlc⁺ progenitor clusters develop.

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.

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.

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.

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.

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.