Expression of Wnt genes during mouse preimplantation development

Susan Lloyda,b, Tom P. Flemingc, Jane E. Collinsa,*

aSchool of Medicine, Mailpoint 813, Southampton General Hospital, Southampton SO16 6YD, UKbLeukaemia Research, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK

cSchool of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO167 PX, UK

Received 24 September 2002; received in revised form 15 November 2002; accepted 10 February 2003

Abstract

Pattern formation in the mouse preimplantation embryo is tightly regulated and essential for successful development. Wnt genes are known

to regulate cell interactions and cell fate in invertebrates and vertebrates and, therefore, may play a role in the specification of cell lineages

and cellular interactions that occur in preimplantation development. Using degenerate primers based on conserved protein sequences in Wnt

coding regions, we have found evidence for Wnt gene expression at the blastocyst stage of mouse preimplantation development. We have

identified sequences encoding Wnts3a and 4 and confirmed that these are present as transcripts in early development by using reverse

transcriptase-polymerase chain reaction (RT-PCR) with specific primers located in the 50 half of these Wnt genes. Studies on the timing of

expression showed that Wnt3a transcripts were present in 2-cell embryos which may represent maternally or embryonically derived

transcripts since the major transition of maternal to zygotic gene expression occurs during the late 2-cell stage. Both Wnt3a and 4 transcripts

were detected in some precompact 4/8-cell stages with consistent expression detected in all compact 8-, 16-cell and blastocyst stages. To our

knowledge, expression of Wnt genes has not been previously described at such an early stage of mammalian development.

q 2003 Elsevier Science B.V. All rights reserved.

Keywords: Wnt genes; Blastocyst stage; Zygotic gene expression; preimplantation

1. Results and discussion

Mouse preimplantation development results in the

formation of a blastocyst with an outer polarised epithelium,

the trophectoderm and an inner cell mass, from which the

foetus will develop (Fleming et al., 2001; Collins and

Fleming, 1995b). Little is known about the mechanisms

regulating these fundamental processes of pattern

formation.

Wnt proteins form a family of conserved, secreted

molecules that regulate cell-to-cell interactions during

embryogenesis and postnatal development from nematodes

to mammals (Wodarz and Nusse, 1998; Hecht and Kemler,

2000; Kuhl et al., 2000; Huelsken and Birchmeier, 2001;

Miller, 2001). To date at least 19 Wnt genes have been

discovered in mouse and vertebrates, with seven in

invertebrates (Gavin et al., 1990; Bergstein et al., 1997;

Sidow, 1992; Graba et al., 1995). Wnt proteins show diverse

functional effects between cell types that may be exerted via

distinct intracellular signalling pathways (Moon et al., 1997;

Brandon et al., 2000; Wilson et al., 2001). The onset of

epithelial differentiation in the mouse early embryo, with

activation of E-cadherin adhesion and the acquisition of cell

polarity, bears strong similarities to other mesenchymal

epithelial transitions known to involve Wnt signalling. In

epithelial transformation of metanephric mesenchyme

(Stark et al., 1994; Yoshino et al., 2001; Kispert et al.,

1998), simple epithelial bodies are induced in a time frame

similar to that seen in epithelial induction in mouse

blastocyst formation. It is, therefore, surprising that this

important gene family has not been studied in preimplanta-

tion development.

We used a reverse transcriptase-polymerase chain

reaction (RT-PCR) strategy with degenerate oligonucleo-

tide primers to conserved amino acid sequences (Gavin

et al., 1990) to amplify cDNA products from mouse morula

and blastocyst mRNA (Fig. 1). Faint ,400 bps products

were purified and reamplified (Fig. 2) for cloning and

sequencing. Randomly picked cDNA clones containing an

insert were sequenced. Wnts4, 3a and 7a were identified as

1567-133X/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S1567-133X(03)00046-2

Gene Expression Patterns 3 (2003) 309–312

www.elsevier.com/locate/modgep

* Corresponding author. Tel.: þ44-23-80796447; fax: þ44-23-

80795025.

E-mail address: jec3@soton.ac.uk (J.E. Collins).

the most abundantly represented (Table 1). The positioning

of the degenerate primers at the most conserved sites in the

30 half of Wnt gene coding regions leads to the amplification

of highly conserved regions of cDNA that have no introns in

the corresponding genomic DNAs making it possible that

the presence of contaminating genomic DNA could lead to

the amplification of Wnt sequences that are not actually

transcribed in the cells. To exclude the possibility that

genomic sequences were detected in the absence of introns,

nested Wnt primers specific for Wnts3a, 4 and 7a were

designed to amplify the 50 half of mRNA sequences in

regions that span exon splice sites, predicted from BLAST

comparisons of mouse and human Wnt cDNA sequences

with the human genome (Batzoglou et al., 2000). This

approach facilitates the amplification of specific mouse Wnt

mRNAs and allows the identification of mRNA sequences

that are distinguished from genomic DNA sequences.

Primers were tested, using mouse genomic DNA templates

(see Table 2) under the same PCR reaction conditions used

on first strand cDNA, to verify the absence of products (data

not shown). Using this approach, only Wnt3a and 4 mRNA

could then be detected in blastocyst cDNA. Results were

confirmed by sequencing. Wnt7a transcripts were not

detected using three sets of nested primers (data not shown).

Timing of expression was then assessed from 2-cell to

blastocyst (Fig. 3). Wnt expression was monitored in

three or more mRNA samples per stage. Wnt4 was absent

in 2-cell embryos but the onset of expression was detected in

4/8-cell stages with strong expression in compact 8- and

16-cell embryos and in blastocysts. Wnt3a was detected in

2-cell embryos, less strongly in 4/8-cell stages, with robust

expression in compact 8-, 16-cell and early blastocysts. The

source of the Wnt 3a transcripts in 2-cell embryos may be

maternal or embryonic as the major maternal to zygotic

transcriptional transition occurs at the late 2-cell stage

(Schultz, 2002). Further analysis of oocyte mRNA com-

bined with studies of embryos undergoing mRNA synthesis

inhibition during the 2- to 4-cell transition would be

required to adequately differentiate between these

possibilities.

The demonstration that Wnt3a and Wnt4 transcripts are

detected just prior to the phase where the first epithelial cells

differentiate de novo suggests that Wnts have a role in

compaction and preimplantation development, although

clearly, we cannot exclude the possibilities that transcrip-

tion is occurring in the absence of translation (Kidder and

McLachlin, 1985; Sheth et al., 1997) or that embryo

isolation and culture might lead to differences in transcrip-

tion that do not occur in vivo (Natale et al., 2001). Indeed,

Fig. 1. Schematic diagram representing Wnt family member mRNAs

showing the relative positions of primers used in degenerate and specific

PCR reactions. The degenerate primers were based on the amino acid

codes, shown as single letters, and all possible combinations at positions of

redundancy in the genetic code were included in the degenerate

oligonucleotide mixtures. The specific Wnt3a and Wnt4 primers were

used in nested PCR reactions in combinations of primers 1 with 2 in the first

reaction and primers 3 with 4 in the second reaction. Sequences of these

primers are shown below.

Fig. 2. RT-mediated PCR on morula and blastocyst mRNA using consensus Wnt primers. TBE agarose gels (2%) showing DNA products obtained using

degenerate oligonucleotide mix based on consensus Wnt family sequences on morula (16) and blastocyst (32) cDNA in initial PCR reactions (left-hand gel)

and in PCR re-amplification of purified products (right-hand gel), marker lane (M) 100 bp ladder (Life Technologies).

Table 1

Identity and number of clones sequenced from degenerate PCR reactions

using consensus Wnt primers

Wnt Number of clones

3a 7

4 9

7a 6

10b 1

Miscellaneous non-Wnt 7

S. Lloyd et al. / Gene Expression Patterns 3 (2003) 309–312310

epithelial transformation of metanephric mesenchyme can

be induced both by fibroblasts expressing either Wnt4 or

Wnt3a or indeed other Wnts (Kispert et al., 1998). The

observation that individual Wnt3a and Wnt4 knockouts

develop past implantation (Takada et al., 1994; Lee et al.,

2000; Stark et al., 1994) raises the possibility that Wnt3a and

4 may be functionally interchangeable at this stage of

development or that more Wnts remain to be discovered in

this system. Since Wnts3a and 4 are known to be on different

chromosomes in the mouse, a double Wnt3a/4 knockout

should be feasible and could address the question of

whether Wnt3a and Wnt4 have important roles in pre-

implantation development and whether they are function-

ally interchangeable.

2. Materials and methods

2.1. Amplification and cloning of Wnt genes from mouse

blastocyst mRNA

The zona pellucida removal from embryos was achieved

as described previously (Fleming et al., 1991) just prior

to RNA extraction. Poly (A) þ RNA from five mouse

blastocysts was extracted and reverse transcribed into first

strand cDNA (Collins and Fleming, 1995a). cDNA was

amplified with 400 pmol of forward and reverse degenerate

primer mixes in 100 ml amplification reactions using Vent

polymerase (New England Biolabs). Reactions were

incubated at 958C for 3 min then cycled five times at

958C, 30 s; 468C, 30 s; 728C, 25 s, then 35 cycles of 958C,

30 s; 608C, 30 s; 728C, 25 s. Four hundred base pair

products were excised from gels, purified using Wizard

PCR kits (Promega) and reamplified (2 ml) under the same

reaction conditions using 35 cycles of 958C, 30 s; 608C,

30 s; 728C, 25 s, in order to obtain more DNA for subse-

quent manipulation. Resulting products were cloned into

pGEM T Easy vectors (Promega) and sequenced using Big

Dye reagents on ABI 377 sequencers (Applied Biosystems).

2.2. Production of staged embryos and expression analysis

of Wnts3a and 4 in preimplantation development

Preimplantation embryos at the early 2-cell stage were

obtained and cultured as described in detail (Collins et al.,

1995). Embryos were selected at 2-cell, precompact

4/8-cell, compact 8-cell, 16-cell and blastocyst, 32- to

64-cells. The zona pellucidae were removed and Poly (A) þ

RNA prepared as described above. Specific nested PCR

primers for Wnt3a, 4 and 7a (Fig. 1) were used at 50 pmol

with cDNAs for first and second stage reactions: 958C,

3 min then cycled twice at 958C, 30 s; 648C, 30 s; 728C, 25 s

followed by two similar cycles at each annealing tem-

perature of 638C, 628C, 618C, 608C, 598C, 588C, 578C, 568C,

with 22 cycles, annealing at 558C. E-cadherin primers

were used to verify mRNA integrity with forward,

Table 2

Sizes of specific Wnt cDNA products generated with inner nested primers

compared with the predicted corresponding genomic DNA sequences

Wnt gene cDNA product size with

innermost primers

Predicted genomic DNA

product size

Wnt4 401 bps .8438 bps

Wnt3a 389 bps ,43607 bps

Wnt7a 353 bps ,20,141 bps

Fig. 3. Expression of Wnt3a and 4 in mouse early embryos. Detection of transcripts for Wnts3a and 4 in staged mouse preimplantation embryos. Lanes: outer

left- and right-hand lanes, 100 bp ladder; 2, 2-cell embryos; 4/8, 4- to 8-cell precompacted embryos, C8, 8-cell compacted embryos; 16, 16-cell embryos; 32,

32- to 64-cell blastocysts; C, control with no mRNA to exclude contamination. E-cadherin transcripts were detected in control aliquots of embryo cDNA to

check for mRNA integrity and are shown on the lower gels directly below the corresponding experimental samples.

S. Lloyd et al. / Gene Expression Patterns 3 (2003) 309–312 311

CCATTTTCACGCGCGCTG and reverse CGCGAGCTT-

GAGATGGAT primers to give a 396 bp product.

Acknowledgements

This work was funded by the Medical Research Council,

UK and The Wellcome Trust. We thank Kate Hayes for

assistance in preparing the figures.

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