DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 4159 Development 137, 4159-4169 (2010) doi:10.1242/dev.056630 © 2010. Published by The Company of Biologists Ltd Initiation of trophectoderm lineage specification in mouse embryos is independent of Cdx2 Guangming Wu1, Luca Gentile1, Takuya Fuchikami2, Julien Sutter1, Katherina Psathaki1, Telma C. Esteves1, Marcos J. Araúzo-Bravo1, Claudia Ortmeier1, Gaby Verberk1, Kuniya Abe2 and Hans R. Schöler1,3,* SUMMARY The separation of the first two lineages – trophectoderm (TE) and inner cell mass (ICM) – is a crucial event in the development of the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2, the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation, but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated. Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a ‘switch’ regulator to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation of ICM/TE lineage separation. KEY WORDS: Cdx2, Trophectoderm, Lineage specification, Preimplantation mouse embryos INTRODUCTION and in establishing anteroposterior patterning of the intestine The first visible cell lineage split in the development of the (Silberg et al., 2000). Dysregulation of Cdx2 expression has been mammalian embryo occurs during blastulation. The cells of the found in intestinal metaplasia and carcinomas (da Costa et al., inner part of the blastocyst, called the inner cell mass (ICM), are 1999; Bai et al., 2002; Eda et al., 2003). In colon cancer cells, for pluripotent and eventually give rise to the embryo proper and to the example, oncogenic ras downregulates Cdx2 expression by extra-embryonic tissues. By contrast, the cells of the outer layer activating protein kinase C (PKC) pathway signaling and by differentiate into an epithelium, called the trophectoderm (TE), reducing activity of the Cdx2 promoter AP-1 site through changes which subsequently develops into the placenta. To date, a full in the relative expression of c-June/c-Fos (Lorentz et al., 1999); by understanding has been lacking of the molecular mechanisms that contrast, in the embryo, ras-MAPK signaling activates Cdx2 underlie the fate decision for the initial totipotent cells of the expression (Lu et al., 2008). Most importantly, one study found that embryo to become either the TE or ICM lineage. although Cdx2 expression appears to be required for TE cell fate Cdx2 is a class I homeobox transcription factor that belongs to specification in the early mouse embryo, Cdx2 zygotic mutants can the caudal-related homeobox gene family, which comprises still initiate TE differentiation and blastulation (Strumpf et al., mammalian homologs of Drosophila caudal (James et al., 1994; 2005). Similarly, another study found that Cdx2-deficient embryos, Suh et al., 1994). Cdx family members function as upstream generated from Cdx2 short hairpin (sh) RNA-expressing somatic positive regulators of Hox genes (Lorentz et al., 1997), which are cells using nuclear transfer, failed to implant into the uterus, just purported to confer positional identity to cells along the like Cdx2–/– embryos (Meissner and Jaenisch, 2006). However, anteroposterior body axis and are sequentially activated in a neither study eliminated maternal Cdx2 expression. More recent temporal and spatial manner (Davidson et al., 2003). Loss of Cdx studies have found that Tead4 acts upstream of Cdx2 and is function has been shown to be associated with inactivation of Hox essential for TE specification and blastocyst formation (Yagi et al., gene expression (Davidson et al., 2003). Cdx1 and Cdx2, which are 2007; Nishioka et al., 2008), although a subsequent study regulated by p38 MAPK (Mapk14) (Houde et al., 2001), are contradicted these findings by reporting that maternal Cdx2 is involved in defining the anteroposterior body axis responsible for compaction and TE lineage initiation (Jedrusik et (Chawengsaksophak et al., 1997; Chawengsaksophak et al., 2004) al., 2010). Therefore, although it is well established that Cdx2 is indispensable for the maintenance and proper functioning of the TE lineage, its role in the initiation of TE lineage specification remains obscure. It is therefore imperative to study the full effect of Cdx2 1 Department of Cell and Developmental Biology, Max Planck Institute for Molecular expression in early mammalian embryonic development by Biomedicine, Röntgenstrasse 20, 48149 Münster, Germany. 2BioResource Center, RIKEN Tsukuba Institute, Ibaraki 305-0074, Japan. 3University of Münster, Medical eliminating both maternal and zygotic Cdx2 transcripts. Faculty, Domagkstr. 3, 48149 Münster, Germany. In the present study, we microinjected a robust Cdx2 small interfering (si) RNA duplex into zygotes and metaphase II (MII) *Author for correspondence ([email protected]) oocytes and determined the effects of eliminating both maternal Accepted 11 October 2010 and zygotic expression of Cdx2 on the initiation of ICM/TE lineage DEVELOPMENT 4160 RESEARCH ARTICLE Development 137 (24) separation and cellular differentiation. As Cdx2 acts downstream amplification were custom designed: PF, 5Ј-AACCAGTGGTTGAATA - of Tead4, we also targeted Tead4 with siRNA to further confirm CTAGCAATG-3Ј; PR, 5Ј-CTGCAATGGATGCTGGGATACT-3Ј; and these results. The findings of this study help to better define the probe, 5Ј-6FAM-TTCAGAAGGGCTCAGCAC-MGB-3Ј. Three to six roles played by Cdx2 during early mammalian development. biological replicates were used and each sample was run with three technical replicates; negative controls lacked reverse transcriptase or MATERIALS AND METHODS template. The cycle threshold (CT) values were collected using Applied Embryo culture and microinjection of siCdx2 duplex Biosystems SDS v2.0 software and transferred to a Microsoft Excel Fertilized oocytes were collected from the oviducts of primed B6C3F1 spreadsheet for further relative quantification analysis using the DDCT female mice after mating with CD1 male mice 18 hours post-hCG in M2 method (User Bulletin #2, ABI Prism 7700 Sequence Detection System, medium. Oocytes were cultured in KSOMAA (potassium simplex optimized 1997). Some of the data were presented using the percentage of peak method, which is an adaptation of the C method (Wang et al., 2004). medium plus 19 natural amino acids) (Ho et al., 1995) at 37°C and 5% CO2 DD T in air until microinjection. For MII oocyte microinjection, mature oocytes Immunofluorescent staining of embryos were collected from the oviducts of primed B6C3F1 female mice 14 hours Immunocytochemical staining was performed as described previously with post-hCG in M2 medium, injected with siCdx2, and then fertilized in vitro minor modifications (Strumpf et al., 2005). Briefly, samples were fixed in in modified KSOM (Summers et al., 2000) with epididymal spermatozoa 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X- from adult OG2 male mice. 100 (Sigma-Aldrich, St Louis, MO, USA) for 1 hour, and blocked with 3% We used the online tool BLOCK-iT RNAi Designer to select specific BSA in PBS for 1 hour. Control and Cdx2-deficient embryos were target sequences for siRNA (https://rnaidesigner.invitrogen.com/ processed and examined in parallel. Samples were incubated with the rnaiexpress/). The lyophilized siRNA duplexes (Invitrogen, Karlsruhe, following antibodies: monoclonal mouse anti-Cdx2 IgG (1:100; BioGenex, Germany) were resuspended in 1 ml DEPC-treated water according to the San Ramon, CA, USA), rabbit polyclonal anti-Cdx2 [1:2000; gift from manufacturer’s instructions and stored in single-use aliquots at –20°C. We Felix Beck (Victoria, Australia) and Alexey Tomilin, (Freiburg, Germany)], tested three regular oligonucleotides and three Stealth RNAi rabbit anti-Nanog IgG (1:500; Cosmo Bio Company, Tokyo, Japan), oligonucleotides containing the coding region of Cdx2 (target sequence 5Ј- monoclonal mouse anti-Oct4 IgG (1:200; SC-5279; Santa Cruz Biotech., GCAGTCCCTAGGAAGCCAA-3Ј) in zygotes and selected the most Santa Cruz, CA, USA), rabbit anti-Oct4 IgG [1:2500; generated in our effective duplex (siRNA3) for use in all our experiments. Unless otherwise laboratory (Palmieri et al., 1994)], rat anti-E-cadherin IgG (1:100; specified, a scrambled siRNA duplex was used as control (target sequence ECCD-2; Calbiochem, Darmstadt, Germany), Troma-1 (1:100; DSHB, 5Ј-GCACCCGATAAGCGGTCAA-3Ј). Iowa City, IA, USA) or rabbit anti-ISP1 IgG [1:100; kindly provided by siRNAs were microinjected using an Eppendorf FemtoJet microinjector