Essential functions of the Williams-Beuren syndrome-associated TFII-I genes in embryonic development Badam Enkhmandakha, Aleksandr V. Makeyeva, Lkhamsuren Erdenechimega, Frank H. Ruddleb,1, Nyam-Osor Chimgea, Maria Isabel Tussie-Lunac, Ananda L. Royc, and Dashzeveg Bayarsaihana,1 aDepartment of Reconstructive Sciences, University of Connecticut Health Center, Farmington, CT 06032; 2Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520; and 3Department of Pathology, Tufts University School of Medicine, Boston, MA 02111 Contributed by Francis H. Ruddle, November 14, 2008 (sent for review May 15, 2008) GTF2I and GTF2IRD1 encoding the multifunctional transcription a gene-trap clone carrying the LacZ-neo insertion in the 22nd intron factors TFII-I and BEN are clustered at the 7q11.23 region hemizy- of Gtf2ird1. Three Gtf2i mutant lines were generated carrying gously deleted in Williams-Beuren syndrome (WBS), a complex independent insertions within intron 2 (Gtf2iRR105), intron 3 multisystemic neurodevelopmental disorder. Although the bio- (Gtf2iXE029), and intron 9 (Gtf2i XC455)oftheGtf2i locus (Fig. 1). In chemical properties of TFII-I family transcription factors have been all 4 gene-trap lines, the LacZ-neo insertion was upstream of the studied in depth, little is known about the specialized contribu- functional nuclear localization signal of BEN and TFII-I. The tions of these factors in pathways required for proper embryonic rationale to use these lines was that the ␤-galactosidase–fused development. Here, we show that homozygous loss of either polypeptides are not able to translocate to the nucleus, therefore Gtf2ird1 or Gtf2i function results in multiple phenotypic manifes- creating a KO condition in these mice. The gene-trap clones were tations, including embryonic lethality; brain hemorrhage; and injected into blastocysts to produce mice with the inactivated vasculogenic, craniofacial, and neural tube defects in mice. Further Gtf2ird1 and Gtf2i alleles. All 3 Gtf2i lines produced the same analyses suggest that embryonic lethality may be attributable to embryonic defects, indicating that disruption of the Gtf2i allele defects in yolk sac vasculogenesis and angiogenesis. Microarray causes these phenotypic manifestations. Therefore, in all our fol- data indicate that the Gtf2ird1 homozygous phenotype is mainly lowing studies to characterize Gtf2i, we used embryos mainly ␤ caused by an impairment of the genes involved in the TGF RII/ derived from the XE029 line. The Gtf2ird1XE465 and Gtf2iXE029 lines Alk1/Smad5 signal transduction pathway. The effect of Gtf2i were maintained on a mixed C57BL/6 and 129Sv background. inactivation on this pathway is less prominent, but downregula- Most mice heterozygous for the Gtf2ird1 and Gtf2i alleles ap- tion of the endothelial growth factor receptor-2 gene, resulting in peared to be normal, based on their external appearance and the deterioration of vascular signaling, most likely exacerbates the fertility. F1 heterozygous mice were intercrossed to produce severity of the Gtf2i mutant phenotype. A subset of Gtf2ird1 and Gtf2ird1Ϫ/Ϫ and Gtf2iϪ/Ϫ embryos. No homozygotes were recov- Gtf2i heterozygotes displayed microcephaly, retarded growth, and ered at weaning among 367 and 272 offspring of different het- skeletal and craniofacial defects, therefore showing that haploin- erozygous intercrosses of Gtf2ird1 and Gtf2i, respectively. These sufficiency of TFII-I proteins causes various developmental anom- results indicated that loss of either the Gtf2ird1 or Gtf2i allele causes alies that are often associated with WBS. Ϫ Ϫ Ϫ Ϫ embryonic lethality. Gtf2ird1 / and Gtf2i / embryos appeared to be delayed in development by 12 to 24 h based on their body size embryonic ͉ GTF2I ͉ GTF2IRD1 and die between embryonic day (E) 8.5 and E12.5. A large number of homozygous embryos at E8.5 did not initiate axial turning, he TFII-I transcription factors represent a versatile protein reflecting retarded development. In some mutant embryos, we Tfamily with broad functional activities (1–4). The GTF2IRD1 observed abnormal development of the allantois, which appeared and GTF2I paralogs encoding BEN and TFII-I proteins, respec- swollen and failed to fuse to the chorion (Fig. 2C). At E9.5 and tively, are clustered at the 7q11.23 region hemizygously deleted in E10.5, surviving Gtf2ird1Ϫ/Ϫ and Gtf2iϪ/Ϫ embryos show growth Williams-Beuren syndrome (WBS), a disorder characterized by retardation, dilated pericardial sacs with arrested heart looping, distinctive facial features, mental disability, and growth retardation hypoplasia of branchial arches, and small frontonasal primordia (5, 6). The protein products of the syntenic Gtf2i and Gtf2ird1 on (Fig. 2 B–D). Many embryos at this stage were already resorbed. A murine chromosome 5 share considerable sequence homology few embryos were found with midfacial clefts (Fig. 2 G and H). BIOLOGY within a repeated domain, which is involved in sequence-specific Embryonic hemorrhage of the mutant embryos was apparent at DEVELOPMENTAL DNA-binding properties (7–10). Comparative sequence analysis of E9.5 (Fig. 2D). The incidence and severity of the hemorrhage the TFII-I family members suggests that these genes have evolved increased during development, and at E12.5, over 60% of embryos from a single ancestor via duplication and divergence (7, 11). Our suffered serious bleeding (Fig. 2 K, N–Q). Mutant embryos dis- Gtf2ird1 Gtf2i previous studies revealed that products of and are played varying degrees of intraembryonic bleeding in the head, expressed very early in mouse development (12–14). Although neck, heart, and back areas. Blood vessels in the yolk sac of biochemical properties of these factors have been analyzed over the Ϫ Ϫ Gtf2ird1 / embryos were poorly developed, resulting in an obvi- past few years, little is known about their physiologic role during ously pale yolk sac compared with that of WT littermates (Fig. 2 L embryogenesis. To define the individual function of TFII-I pro- and M). The mutant embryos themselves were pale and showed teins, we have generated mouse lines mutant for Gtf2ird1 and Gtf2i alleles and provide evidence that TFII-I family factors are crucial in various aspects of mouse development. Author contributions: D.B. designed research; B.E., A.V.M., L.E., N.-O.C., and M.I.T.-L. performed research; B.E., A.V.M., F.H.R., and D.B. analyzed data; and B.E., A.V.M., F.H.R., Results and Discussion A.L.R., and D.B. wrote the paper. ؊ ؊ ؊ ؊ Multiple Embryonic Defects in Gtf2ird1 / and Gtf2i / Embryos. To The authors declare no conflict of interest. elucidate the biological functions of Gtf2ird1 and Gtf2i during 1To whom correspondence should be addressed. E-mail: [email protected] or mammalian embryonic development, we generated 4 mutant [email protected]. mouse lines. One line, designated as Gtf2ird1XE465, was derived from © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811531106 PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 ͉ 181–186 Downloaded by guest on September 28, 2021 EcoR V 1 kb Gtf2i XE029R1 Sca I Geo R 735 Amp bp FRT loxP Sca I XE029F Sca I Sca I Sca I 2 3 TF2IF2 TF2IR2 4 5 6 7 8 8a 9 Fig. 1. Inactivation of Gtf2ird1 and Gtf2i using the gene-trap strategy (http:/www.genetrap.org/). Mu- 6.2 kb tant mice were produced from gene-trap lines carrying 1 2 3 4 M bp the ␤-Geo insertion within intron 22 (clone XE465 XE029 EcoR V - 500 Gtf2ird1 ) or intron 3 (Gtf2i ). Geo, lacZ-neo XF06 Sca I Gtf2ird1 insertion; NLS, nuclear localization signal. Animals Geo R - 300 were genotyped by PCR with primers specific for Gtf2i 1.13 kb Amp FRT - 200 (TF2IF2/TF2IR2 and XE029F/XE029R1) and Gtf2ird1 loxP (F11/BEN22R/XF06). The WT Gtf2i allele gives a 437-bp band (line 1), whereas the Gtf2iXE029 allele displays a Sca I EcoR V Sca I Sca I EcoR V 200-bp product (line 2). Similarly, Gtf2ird1-specific primers amplify a 479-bp fragment in WT DNA (line 3) XE465 22 F11 BEN22R 23 24 25 26 27 28 and an additional 320-bp band from the Gtf2ird1 gene-trapped allele (line 4). Positions of primers used 5.9 kb in PCR are shown in the diagram. arrested growth at E9.5, suggesting the possibility of defective 10% of Gtf2iϩ/Ϫ mice were significantly smaller than their WT hematopoiesis in both the embryo body and the yolk sac attribut- littermates despite being viable and fertile (Fig. 4 C and D). The able to defects in blood vessel formation. body weight was 65–75% of the normal weight of adult mice. To An open anterior neural tube (exencephaly) was displayed by evaluate the growth potential of newborn pups, we analyzed WT Ϸ15% of Gtf2iϩ/Ϫ and 2% of Gtf2ird1ϩ/Ϫ mice (Fig. 4B). In and Gtf2ird1ϩ/Ϫ mice over several months. Heterozygotes grew homozygous embryos, this number is larger: 60% of Gtf2iϪ/Ϫ significantly slower than control pups, indicating poor growth rate embryos have exencephaly compared with 9% of Gtf2ird1Ϫ/Ϫ (not shown). We also observed pigmentation defects in some embryos (Fig. 2 C, F, I–K). Normally, the neural tube starts to close heterozygous embryos, albeit at a low frequency. The lack of along the ventral midline around E8.5–9.0 and is completed by E9.5. pigment was observed as white patches of variable size on the belly This process of neurulation is a complex task, which involves cell (Fig. 4E). migration as well as extensive neuroectoderm proliferation and/or Among other abnormalities in Gtf2ird1ϩ/Ϫ animals, we observed cell death. At E9.5, either Gtf2i or Gtf2ird1 homozygotes displayed hydrocephalus and kyphosis, a pronounced arched spine (Fig. 4C). open and everted cranial neural folds. In most cases, closure defects The skeletal staining confirmed that the dorsal ‘‘hump’’ of het- occurred at the forebrain/midbrain boundary and extended ros- erozygous animals resulted from an increased backward curvature trally to the anterior neuropore, caudally to the cervical/hindbrain of the spine (Fig.