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. 2019 Dec 29;12(1):80.
doi: 10.3390/cancers12010080.

Loss of Ing3 Expression Results in Growth Retardation and Embryonic Death

Dieter Fink  1 , Tienyin Yau  1 , Arash Nabbi  2 , Bettina Wagner  1 , Christine Wagner  3 , Shiting Misaki Hu  1 , Viktor Lang  1 , Stephan Handschuh  4 , Karl Riabowol  2 , Thomas Rülicke  1
Affiliations

Loss of Ing3 Expression Results in Growth Retardation and Embryonic Death

Dieter Fink et al. Cancers (Basel). .

Abstract

The ING3 candidate tumour suppressor belongs to a family of histone modifying proteins involved in regulating cell proliferation, senescence, apoptosis, chromatin remodeling, and DNA repair. It is a stoichiometric member of the minimal NuA4 histone acetyl transferase (HAT) complex consisting of EAF6, EPC1, ING3, and TIP60. This complex is responsible for the transcription of an essential cascade of genes involved in embryonic development and in tumour suppression. ING3 has been linked to head and neck and hepatocellular cancers, although its status as a tumour suppressor has not been well established. Recent studies suggest a pro-metastasis role in prostate cancer progression. Here, we describe a transgenic mouse strain with insertional mutation of an UbC-mCherry expression cassette into the endogenous Ing3 locus, resulting in the disruption of ING3 protein expression. Homozygous mutants are embryonically lethal, display growth retardation, and severe developmental disorders. At embryonic day (E) 10.5, the last time point viable homozygous embryos were found, they were approximately half the size of heterozygous mice that develop normally. µCT analysis revealed a developmental defect in neural tube closure, resulting in the failure of formation of closed primary brain vesicles in homozygous mid-gestation embryos. This is consistent with high ING3 expression levels in the embryonic brains of heterozygous and wild type mice and its lack in homozygous mutant embryos that show a lack of ectodermal differentiation. Our data provide direct evidence that ING3 is an essential factor for normal embryonic development and that it plays a fundamental role in prenatal brain formation.

Keywords: ING Proteins; Ing3; embryonic lethal; growth retardation; insertional mutation.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Figure 1
Figure 1
Quantification of overall fluorescence, schematic view of UbC-mCherry integration site, and embryo size. (A) Overall fluorescence of wild type (+/+; n = 4) and hemizygous (+/T; n = 8) UbC-mCherry mice, and hemizygous (+/T; n = 4) and homozygous (T/T; n = 4) CAG-mCherry mice. (B) Integration site of the UbC-mCherry cassette at 6qA3.1 disrupting the Ing3 locus. (C) Embryo size of wild type (+/+; n = 9), heterozygous (+/T; n = 19), and homozygous (T/T; n = 8) Ing3 insertional mutants at E10.5.
Figure 2
Figure 2
Protein expression in whole E10.5 embryos and the developing brain of E10.5 embryos. (A) Western blot analysis for ING3 of wild type (+/+; n = 1), heterozygous (+/T; n = 1), and homozygous (T/T; n = 2) whole embryos and intensity ratio readings ING3/Tubulin. (B) Immunohistochemisty for ING3 of wild type (+/+; n = 2), heterozygous (+/T; n = 2), and homozygous (T/T; n = 1) embryos. A representative section of the developing brain is shown and indicated by arrows. Magnification 40×.
Figure 2
Figure 2
Protein expression in whole E10.5 embryos and the developing brain of E10.5 embryos. (A) Western blot analysis for ING3 of wild type (+/+; n = 1), heterozygous (+/T; n = 1), and homozygous (T/T; n = 2) whole embryos and intensity ratio readings ING3/Tubulin. (B) Immunohistochemisty for ING3 of wild type (+/+; n = 2), heterozygous (+/T; n = 2), and homozygous (T/T; n = 1) embryos. A representative section of the developing brain is shown and indicated by arrows. Magnification 40×.
Figure 3
Figure 3
µCT analysis of E10.5 embryos. Volume renderings show lateral view of (A) wild type (+/+; n = 4), (B) heterozygous (+/T; n = 4), and (C) homozygous (T/T; n = 3) embryos. Virtual µCT sections of (D,E,F,G) wild type, (H,I,J,K) heterozygous, and (L,M,N,O) homozygous embryos. Note that in (A) the tail bud was clipped unintentionally during specimen preparation and in (B) the hindlimb bud and tail bud are located behind the embryo head and thus are hidden in the image. (1, 2, 3: branchial bars 1–3; bb: brain bulges; ed: endolymphatic duct; fb: forelimb bud; hb: hindlimb bud; he: heart; lp: lens pit; me: mesencephalon; oc: optical cup; op: olfactory pit; os: optic stalk; ov: otic vesicle; rh: rhombencephalon; Rp: Rathke’s pouch; rs: retinal sheet; te: telencephalon).
Figure 4
Figure 4
Reconstitution of ING3 by ubiquitous expression of Ing3-P2A-eGFP. (A) Schematic view of the Ing3 transposon construct. (B) In vivo imaging showing mCherry expression of different genotypes in a representative litter of a UbC-mCherry, CAG-Ing3-P2A-eGFP double mutant bred to a hemizygous UbC-mCherry mouse. (C) mCherry and eGFP expression with corresponding genotypes of UbC-mCherry and CAG-Ing3-P2A-eGFP transgenic pups (homozygous (T/T), hemizyous (+/T) and wild type (+/+)). Total numbers of animals per genotype are indicated in the figure. Position of genotyping primers are indicated by arrows.
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