Loss of mouse Ikbkap, a subunit of elongator, leads to transcriptional deficits and embryonic lethality that can be rescued by human IKBKAP

Abstract

Familial dysautonomia (FD), a devastating hereditary sensory and autonomic neuropathy, results from an intronic mutation in the IKBKAP gene that disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP) in the nervous system. To better understand the roles of IKAP in vivo, an Ikbkap knockout mouse model was created. Results from our study show that ablating Ikbkap leads to embryonic lethality, with no homozygous Ikbkap knockout (Ikbkap(-)(/)(-)) embryos surviving beyond 12.5 days postcoitum. Morphological analyses of the Ikbkap(-)(/)(-) conceptus at different stages revealed abnormalities in both the visceral yolk sac and the embryo, including stunted extraembryonic blood vessel formation, delayed entry into midgastrulation, disoriented dorsal primitive neural alignment, and failure to establish the embryonic vascular system. Further, we demonstrate downregulation of several genes that are important for neurulation and vascular development in the Ikbkap(-)(/)(-) embryos and show that this correlates with a defect in transcriptional elongation-coupled histone acetylation. Finally, we show that the embryonic lethality resulting from Ikbkap ablation can be rescued by a human IKBKAP transgene. For the first time, we demonstrate that IKAP is crucial for both vascular and neural development during embryogenesis and that protein function is conserved between mouse and human.

FIG. 1.

Gene targeting strategy and Ikbkap expression. (A) Schematic of the wild-type and knockout Ikbkap alleles. The blue cassette (β-geo) represents the vector containing β-galactosidase, neomycin phosphotransferase II, and stop codons that is inserted into intron 9. The insertion creates a fusion transcript containing the exons upstream of the insertion joined to the β-geo marker, as illustrated by the red lines. (B) PCR genotyping results of genomic DNA from embryonic day 9.5 samples. Lanes 1, 2, and 3 represent the wild-type (Ikbkap^+/+), heterozygous (Ikbkap^+/−), and homozygous (Ikbkap^−/−) genotypes, respectively. wt, wild-type fragment (454 bp); ko, knockout fragment (244 bp). (C) The relative amounts of Ikbkap transcripts expressed in embryos with different genotypes at 8.5 dpc as demonstrated by quantitative RT-PCR. The error bars indicate standard deviations. (D and E) X-Gal staining of whole-mount Ikbkap^+/− embryos at 8.5 and 11.5 dpc, respectively. The arrowheads in panel D point to the ventral and dorsal neural tubes; note that the primitive hindbrain region shows higher positive reactivity. The arrowheads in panel E point to the hindbrain and dorsal ganglia. A, anterior; P, posterior. Scale bars, 1 mm.

FIG. 2.

Appearance of Ikbkap^+/+ and Ikbkap^−/− extraembryonic components at different stages. Shown is the morphology of the Ikbkap^+/+ and Ikbkap^−/− conceptus at 6.5 to 12.5 dpc under a dissection microscope. (A) At 6.5 dpc, no gross abnormalities are found in the Ikbkap^−/− conceptus compared to a wild-type control. epc, ectoplacental cone. (B) At 7.5 dpc, the Ikbkap^−/− epc and visceral yolk sac (vys), as well as the embryo inside, are smaller than those found in Ikbkap^+/+ controls. Note that at this stage the blood islands are readily observable in the Ikbkap^+/+ vys; however, no corresponding architectures are found in the Ikbkap^−/− vys. (C) At 9.5 dpc, the Ikbkap^−/− vys, as well as the embryo inside, are smaller than those found in the Ikbkap^+/+ control. At this stage, the blood vessels can easily be identified in the Ikbkap^+/+ vys; however, in the Ikbkap^−/− vys, only the primary capillary plexus is observed (inset). (D) At 12.5 dpc, the placenta can be found in both genotypes; in contrast, no embryo can be seen inside the Ikbkap^−/− vys. P, placenta. Scale bars, 0.5 mm (A and B), 1 mm (C), and 2.5 mm (D).

FIG. 3.

Gene expression patterns of the Ikbkap^+/+ and Ikbkap^−/− visceral yolk sac at 8.5 dpc. Shown is semiquantitative RT-PCR analysis of marker genes for vasculogenesis and angiogenesis at 8.5 dpc in the Ikbkap^+/+ and Ikbkap^−/− visceral yolk sacs. The values represent the integrated density values of the bands relative to the average of the Ikbkap^+/+ expression. The names of the genes examined and the genotypes of samples are indicated. The samples in lane I and lane III, as well as lane II and lane IV, were harvested from littermates.

FIG. 4.

Appearance of Ikbkap^+/+ and Ikbkap^−/− embryos at different embryonic stages. Shown are morphological phenotypes of the Ikbkap^+/+ and Ikbkap^−/− embryos from 7.5 to 10 dpc. At 7.5 dpc, the Ikbkap^+/+ embryo undergoes gastrulation and formation of primitive organized structures, such as allantois and head folds (A, inset); however, the Ikbkap^−/− embryos appeared to be arrested at the late primitive streak stage, and this feature persisted to 8.5 dpc in the Ikbkap^−/− embryo (B). At 8.5 and 9.5 dpc, closure of the anterior neural tube (arrowheads in panels B and C) and body rotation could be observed in wild-type embryos (B and C); in contrast, the Ikbkap^−/− embryos lacked these developmental features (B and C). At 9.5 and 10 dpc, the characteristics of the midgastrulation stage, such as allantois, primitive heart, and head fold (C, inset, and D), could be found in the Ikbkap^−/− embryo; however, no blood vessels were observed compared with the Ikbkap^+/+ control (D). Scale bars, 0.5 mm; in panel D, the scale bar adjacent to the Ikbkap^+/+ embryo is 1 mm.

FIG. 5.

Appearance of Ikbkap^+/+ and Ikbkap^−/− embryos at 10.5 dpc. Shown is a morphological analysis of the Ikbkap^+/+ and Ikbkap^−/− embryos at 10.5 dpc. (A) At this stage, in the Ikbkap^+/+ embryo, structures of the forebrain and hindbrain can be identified from ventral and dorsal aspects of the embryo, and the forelimb and hindlimb buds can be seen. (B) In contrast, at 10.5 dpc, incomplete turning is observed in the Ikbkap^−/− embryo. No corresponding primitive brain structures or limb buds can be located; however, the anterior neural tube is closed from the ventral view, and the unsmooth zipper-like posterior neural tube can be found. Scale bars, 1 mm (A) and 0.5 mm (B).

FIG. 6.

Gene expression patterns of the Ikbkap^+/+ and Ikbkap^−/− embryos at 8.5 dpc. Shown is RT-PCR analysis of genes using RNA isolated from Ikbkap^+/+ and Ikbkap^−/− embryos. The names of the genes examined and genotypes of samples are indicated. Note that the samples in lane I and lane III, as well as lane II and lane IV, were harvested from littermates.

FIG. 7.

Transcriptional analysis of Ctnnb1, Bmp4, Vegfa, and Smad2 in Ikbkap^+/+ and Ikbkap^−/− embryos. (A) Schematic representation of the genes investigated by ChIP assay. Exons are depicted by boxes; the closed boxes indicate the localization of the amplicons. The numbers (kb) indicate the positions of these amplicons relative to the 5′ sites of genes. (B) The acetylation status of histone H3 in the transcribed regions of Ctnnb1, Bmp4, Vegfa, and Smad2 was estimated using an acetyl-histone H3 ChIP assay. Note that Ctnnb1 exon 8, Bmp4 exon 2, and Vegfa exon 3 were not pulled down with anti-acetyl-histone H3 antibody in Ikbkap^−/− embryos. The genes tested, the genotypes, and the locations (E, exon) of amplicons are indicated. α-Acetyl H3, anti-acetyl-histone H3 antibody; No Ab, no antibody control.

FIG. 8.

Appearance of Ikbkap^−/− mice with a human wild-type IKBKAP transgene. (A) The arrowheads point to Ikbkap null mice with the human wild-type IKBKAP transgene. No significant phenotypic differences were observed compared to heterozygous littermates. (B) PCR genotyping results of genomic DNA from tail snips with dedicated primers. Lane 1 (from left), Ikbkap^+/−genotype; lanes 2 and 3, Ikbkap1^−/− genotype. The mice represented by lanes 2 and 3 were positive for carrying the human transgene. wt, wild-type fragment; ko, knockout fragment; TG, human IKBKAP transgene. (C) Western blot result using the human-specific IKAP antibody to confirm the presence of the human IKBKAP protein, IKAP, in mouse brains from different genotypes. Lane 1, positive control of nuclear extracts from HeLa cells, which are positive for IKAP expression; lane 2, control mouse (no human IKBKAP [WT] transgene); lane 3, Ikbkap^+/+ mouse with human WT transgene; lane 4, Ikbkap^−/− mouse with human WT transgene; lane 5, Ikbkap^−/− mouse with human FD transgene. Arrowhead 150-kDa position.