QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 October 2007
doi: 10.1242/dev.010223

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Development Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yagi, R. Articles by Buonanno, A. Search for Related Content PubMed PubMed Citation Articles by Yagi, R. Articles by Buonanno, A. Social Bookmarking                         What's this?

Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development

Rieko Yagi^1,*, Matthew J. Kohn^1,*, Irina Karavanova^2,*, Kotaro J. Kaneko¹, Detlef Vullhorst², Melvin L. DePamphilis¹ and Andres Buonanno^2,

¹ Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, National Institutes of Health, 6 Center Drive, Bethesda, MD 20892-2753, USA.
² Section on Molecular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, 35 Lincoln Drive, Bethesda, MD 20892-3714, USA.

View larger version (45K): [in this window] [in a new window]   Fig. 1. Generation of conditional and full Tead4 knockout mice. (A) Exon 2 of the mouse Tead4 locus (black boxes) was targeted for homologous recombination. Indicated are the positions of the left arm probe (LAP), right-arm probe (RAP), and binding sites for PCR primers used for genotyping (Table 1), and restriction sites used for ES cell screening and for the insertion of loxP sites (open arrows in targeting vector). After homologous recombination, exon 2 is flanked by loxP sites in the conditional Tead4loxP allele (Cond.). Following Cre-mediated recombination, the Tead4 knockout (KO) allele lacks exon 2. Genomic DNA from ES cells (B) and tail snips (C) was subjected to Southern blotting-hybridization with the LAP. (D,E) Genotypes were routinely confirmed by PCR analysis of tail DNA obtained from the progeny of matings between Tead4+/- mice showing the absence of Tead4-/- offspring. Primers: P1, P2, P4 in D and TEAD4.20 (20F), TEAD4.27 (27R), TEAD4.28 (28R) in E. (F) PCR-based genotyping, using primers P1, P2, and P3, of F2 animals produced from mating heterozygotes conditional (Tead4+/lox) animals shows recovery of wild-type, heterozygous and homozygous Tead4 conditional offspring. W, wild type; L, loxP; M, mutant.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Tead4 is expressed in preimplantation embryos and trophoblast cell lines. (A,B) Real-time RT-PCR was used to quantify the levels of Tead4 mRNA in unfertilized eggs, 1-cell embryos (1-C), 2-cell embryos (2-C), 8-cell embryos (8-C), morulae (M) and blastocysts (B) (A), as well as in trophoblast stem (TS) cells, trophoblast giant (TG) cells, embryonic stem (ES) cells and embryoid bodies (EB) (B). Each sample was internally normalized to Gapdh mRNA. Gapdh mRNA level per egg or embryo in A and Gapdh mRNA level per cell in B were essentially constant. Error bars represent the s.e.m. of three independent assays. (C,D) In situ hybridization was used to detect Tead4 transcripts at E6.5. Bright-field (C) and dark-field (D) images of an embryo in utero. Tead4 is broadly expressed in all extraembryonic layers and deciduum, but highest levels are in extra-embryonic (Ex) trophoblast cells of the ectoplacental cone (EPC, outlined by a dashed line). No expression was observed in the embryonic germ layers (Emb).

View larger version (44K): [in this window] [in a new window]   Fig. 3. TEAD4 is required for expression of some genes during preimplantation development, but not others. (A) Embryos from Tead4+/- heterozygous intercrosses were collected either at E2.5 or E3.5 and immunostained with anti-CDX2 antibody (green). DNA was labeled with DAPI (blue), and embryos were photographed under phase contrast optics (Phase). Confocal images of DNA and CDX2 stains were merged. Although the top panel appears to show an 8-cell embryo, there are four more nuclei present outside the focal planes shown. Embryos were genotyped after imaging. Wild-type and Tead4 heterozyotes were morphologically indistinguishable, so only wild-type and Tead4 knockout embryos are shown (total number of embryos examined: Tead4+/+, Tead4+/-: 13 for E.2.5 and 14 for E3.5; Tead4-/-: 3 at E2.5 and 4 at E3.5). (B) Embryos from Tead4+/- heterozygous intercrosses were collected at E3.5 and stained for OCT4 protein (green) as described in A. Typical examples are shown of Tead4+/+ blastocysts (n=16), and of Tead4-/- embryos (n=4). (C) Total RNA was isolated from a single E3.5 blastocyst and a single E3.5 abnormal morula, and RT-PCR was used to detect expression of the Tead4, Cdx2, Eomes, Fgfr2 and Oct4 genes. ß-actin RT-PCR products are shown as a reference.

View larger version (48K): [in this window] [in a new window]   Fig. 4. TEAD4 is required for the formation of a blastocoel cavity, but not through regulation of cadherin 1 (Cdh1). (A) E3.5 embryos were treated as in Fig. 3, except that they were immunostained with anti-CDH1 antibody (green). (B) Schematic representation of the relationship between transcription factor TEAD4 and other genes and events in the establishment of the trophectoderm lineage. Upward and downward pointing arrows indicate increased and decreased expression, respectively.

View larger version (126K): [in this window] [in a new window]   Fig. 5. Tead4-/- embryos appear abnormal at E3.5 and fail to form blastocyst outgrowths in vitro. E3.5 embryos from heterozygous matings were collected and cultured in gelatin-treated tissue culture dishes. Tead4+/+ and Tead4+/- embryos appear as normal blastocysts with inner cell masses (icm), trophectoderm (te), blastocoel cavities (bc) and zonae pellucidae (zp), whereas Tead4-/- embryos appear as abnormal morulae. After 5 days in culture, the embryos and their outgrowths (tg, trophoblast giant cells) were collected for genotyping.

View larger version (48K): [in this window] [in a new window]   Fig. 6. Tead4-/- ES cells appear similar to and express the same markers as wild-type cells. (A) Tead4+/+ and Tead4-/- ES cells generated from E2.5 embryos at 10x and 40x magnification. (B) ES cells derived from embryos produced from Tead4 heterozygous matings were genotyped for Tead4. (C) Semi-quantitative RT-PCR of RNA from Tead4+/+ and Tead4-/- ES cells for markers of pluripotentcy including Oct4, Fgf4, Nanog, Sox2 and Rex1, and differentiation [brachyury (T) at 30 cycles], with Gapdh as a reference. (D) Semi-quantitative RT-PCR analysis of embryoid bodies generated from Tead4+/+ and Tead4-/- ES cells at days 0, 1, 2, 4 and 5. Differentiation markers include Rex1 for undifferentiated ES cells, brachyury (T) (at 35 cycles) for mesoderm, Hnf4a and Gata6 for endoderm, and Fgf5 for ectoderm, with Gapdh as a reference control. W, wild type; M, mutant.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Tead4-/-;Meox2-Cre/+ mice were viable despite the absence of Tead4 mRNA. (A) PCR genotyping of mouse tail samples from matings of Tead4lox/lox and Tead4+/-;Meox2-Cre+ animals. (B) Analysis of Tead4 mRNA expression in major tissues isolated from wild-type and Tead4-/-;Meox2-Cre+ mice by RT-PCR. The Tead4 PCR was performed using 37 cycles with Gapdh as a reference (at 35 cycles). W, wild type; L, loxP; M, mutant.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter