Pediatric/Congenital/DevelopmentalTfap2b mutation in mice results in patent ductus arteriosus and renal malformation
Introduction
The transcription factor TFAP-2 beta is a member of the TFAP2 family and is involved in embryonic development and cell cycle regulation; it is enriched in the neural crest and exhibits dynamic expression related to the migration of neural crest cells.1, 2 Similar to other members of the TFAP2 family, the TFAP2 protein consists of a variable transactivation domain at the N-terminus and a highly conserved helix-span-helix dimerization domain for DNA binding at the C-terminus.3, 4, 5
TFAP2B mutations are associated with Char syndrome, a disorder characterized by patent ductus arteriosus (PDA), an unusual facial appearance, and abnormalities of the fingers in humans.6, 7, 8 Khetyar et al.9 reported that a single-base substitution in exon 3 of TFAP2B causes familial isolated PDA, without craniofacial or finger abnormalities. TFAP2B point mutations may be risk factors for nonsyndromic PDA. In mice, Tfap2b is not only expressed in distal tubular epithelial cells in late embryonic stages but is also specifically expressed in the ductus smooth muscle tissues. Moreover, Tfap2b is involved in the maintenance of the highly differentiated state of ductus smooth muscle cells and plays an important role in the survival and maintenance of the collecting duct and tubular epithelial cells.10, 11, 12 Tfap2b-deficient mice die shortly after birth due to congenital polycystic kidney disease or PDA. Moser et al.11 confirmed that polycystic kidney disease from ED16.5 to birth results from excessive apoptosis of renal epithelial cells. The complete loss of Tfap2b also causes heart-limb defects, including PDA and an accessory postaxial digit of the forelimb.12 However, the pathogenesis of these three abnormalities and the mechanism through which Tfap2b interacts with other genes are not clear.
In our previous study, we found a heterozygous c.435_438delCCGG mutation in TFAP2B in all affected individuals of an isolated PDA family lacking facial and finger abnormalities. This mutant gene had a 4-base pair (bp) CCGG deletion in exon 2 and encoded a predicted abnormal 189 amino acid protein owing to a premature stop codon, p.Arg145Argfsx45.13 We also found that the TFAP2B c.435_438delCCGG mutation altered the transcription of the target sequences and only produced a residual protein of about 21 kDa, which lacked a DNA-binding domain, in cultured cells in vitro.14
Based on these previous results, we hypothesize that the c.435_438delCCGG mutation in TFAP2B may be a key mechanism underlying isolated PDA. Accordingly, in this study, we established a mouse model bearing the c.435_438delCCGG mutation (Tfap2bMu/Mu) using CRISPR/Cas9 technology to determine the effects of this mutation at the molecular level. Interestingly, we found that Tfap2bMu/Mu mice showed partially delayed closure of the ductus arteriosus (DA) and renal malformation but did not show limb abnormalities, unlike mice bearing the Tfap2b-null mutation. The effects of this c.435_438delCCGG mutation in our mouse model may be associated with the truncated N-terminal protein, which could disrupt transcription and translation. We think that the residual protein may include an important motif for interactions with target genes involved in the regulation of limb and DA formation.
Section snippets
Tfap2bMu/Mu mouse generation and genotyping
Heterozygous mutant mice with the Tfap2b c.435_438delCCGG mutation (Tfap2bMu/Wt) in exon 2 were produced using CRISPR/Cas9 technology and maintained by Shanghai Biomodel Organism Science and Technology Development (Shanghai, China). The specific guide-RNA (gRNA) and oligo donor DNA were designed as follows: gRNA1, 5′-CTACCACTCTGTCCGCCGGC-3′; gRNA2, 5′-GCAGCAGCACGTCCGGCCGG-3′; and oligo donor DNA sequence,
Establishment and molecular identification of Tfap2bMu/Mu mice
To generate Tfap2bMu/Mu mutant mice, Cas9 mRNA/sgRNAs and c.435_438delCCGG mutant oligos were inserted into exon 2 of mouse Tfap2b and co-injected into zygotes of C57BL/6 mice (Fig. 1A).15, 16, 17 Blastocysts from the injected embryos were transplanted into foster mouse mothers.13, 16, 17 After birth, sequencing analysis and Western blotting confirmed that Tfap2bMu/Mu mice were obtained. Sequencing analysis using primers located upstream of the insertion site indicated that 4-bp CCGG in exon 2
Discussion
Tfap2b is important for the regulation of murine embryonic development.15, 18 The phenotypes associated with the Tfap2b-null mutation established by deleting exon 4 include polycystic kidney disease, PDA, and limb abnormalities.11 Here, we generated a Tfap2bMu/Mu mouse model by introducing a c.435_438delCCGG mutation in exon 2 of Tfap2b to explore the precise role of this frameshift mutation. We found that the Tfap2bMu/Mu mice with the 4-bp deletion in Tfap2b exhibited a phenotype consisting of
Conclusions
The Tfap2b c.435_438delCCGG mutant mice were characterized by delayed closure of the DA and renal abnormalities. The absence of normal Tfap2b protein may disrupt the development of the kidney or other important organs. In addition, Tfap2bMu/Mu mice exhibited disruptions in the development of the ductus smooth muscle, and this may be involved in the pathogenesis of PDA.
Acknowledgment
Authors' contributions: J.W. performed the generation of the mutants, analysis, and microscopy; W.J. and D.Z. performed the experiments sequencing and Western blot; W.W. performed the immunohistochemistry; Y.C. analyzed the data; Z.Z. and F.L. designed the study and contributed analysis tools. J.W. wrote the article.
The authors thank all staff members who participated in our study. They are deeply grateful to the people at the Shanghai Children's Medical Center and to the members of Zhen
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