Novel gene expression patterns along the proximo-distal axis of the mouse embryo before gastrulation.

Abstract

To date, the earliest stage at which the orientation of the anterior-posterior axis in the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5, prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective posterior markers are expressed more proximally, close to the boundary with the extraembryonic region.
To contribute to elucidating the mechanisms underlying the events involved in early patterning of the mouse embryo, we have carried out a microarray screen to identify novel genes that are differentially expressed between the distal and proximal parts of the E5.5 embryo. Secondary screening of resulting candidates by in situ hybridisation at E5.5 and E6.5 revealed novel expression patterns for known and previously uncharacterised genes, including Peg10, Ctsz1, Cubilin, Jarid1b, Ndrg1, Sfmbt2, Gjb5, Talia and Plet1. The previously undescribed gene Talia and recently identified Plet1 are expressed specifically in the distal-most part of the extraembryonic ectoderm, adjacent to the epiblast, and are therefore potential candidates for regulating early patterning events. Talia and the previously described gene XE7 define a gene family highly conserved among metazoans and with a predicted protein structure suggestive of a post-transcriptional regulative function, whilst Plet1 appears to be mammal-specific and of unknown function.
Our approach has allowed us to compare expression between dissected parts of the egg cylinder and has identified multiple genes with novel expression patterns at this developmental stage. These genes are potential candidates for regulating tissue interactions following implantation.

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BioMed Central
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BMC Developmental Biology
Open Access
Research article
Novel gene expression patterns along the proximo-distal axis of the
mouse embryo before gastrulation
Stephen Frankenberg1,2, Lee Smith3, Andy Greenfield3 and
Magdalena Zernicka-Goetz*1
Address: 1The Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, Tennis Court Rd, Cambridge CB2
1QR, UK, 2Inserm, U384, Clermont-Ferrand, F-63001, France and 3MRC Mammalian Genetics Unit, Harwell, Didcot OX11 0RD, UK
Email: Stephen Frankenberg - stephen@frankenberg.com.au; Lee Smith - l.smith@har.mrc.ac.uk; Andy Greenfield - a.greenfield@har.mrc.ac.uk;
Magdalena Zernicka-Goetz* - mzg@mole.bio.cam.ac.uk
* Corresponding author
Abstract
Background: To date, the earliest stage at which the orientation of the anterior-posterior axis in
the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5,
prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective
posterior markers are expressed more proximally, close to the boundary with the extraembryonic
region.
Results: To contribute to elucidating the mechanisms underlying the events involved in early
patterning of the mouse embryo, we have carried out a microarray screen to identify novel genes
that are differentially expressed between the distal and proximal parts of the E5.5 embryo.
Secondary screening of resulting candidates by in situ hybridisation at E5.5 and E6.5 revealed novel
expression patterns for known and previously uncharacterised genes, including Peg10, Ctsz1,
Cubilin, Jarid1b, Ndrg1, Sfmbt2, Gjb5, Talia and Plet1. The previously undescribed gene Talia and
recently identified Plet1 are expressed specifically in the distal-most part of the extraembryonic
ectoderm, adjacent to the epiblast, and are therefore potential candidates for regulating early
patterning events. Talia and the previously described gene XE7 define a gene family highly
conserved among metazoans and with a predicted protein structure suggestive of a post-
transcriptional regulative function, whilst Plet1 appears to be mammal-specific and of unknown
function.
Conclusion: Our approach has allowed us to compare expression between dissected parts of the
egg cylinder and has identified multiple genes with novel expression patterns at this developmental
stage. These genes are potential candidates for regulating tissue interactions following implantation.
Background
At 5.5 days of development (E5.5) the mouse egg cylinder
appears radially symmetrical about its proximo-distal axis
with respect to known molecular markers and to the
arrangement of its three principle tissue layers – epiblast,
extra-embryonic ectoderm and visceral endoderm. How-
ever, shortly after E5.5 the first molecular asymmetries
that determine the anterior-posterior axis begin to
Published: 15 February 2007
BMC Developmental Biology 2007, 7:8 doi:10.1186/1471-213X-7-8
Received: 2 November 2006
Accepted: 15 February 2007
This article is available from: http://www.biomedcentral.com/1471-213X/7/8
© 2007 Frankenberg et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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emerge. These involve movement of a subset of visceral
endoderm cells, anterior visceral endoderm (AVE),
located at the distal tip of the egg cylinder towards the
future anterior side [1-5]. Subsequent to this, molecular
markers with a previously radial distribution near the
embryonic-extra-embryonic boundary become restricted
to the future posterior side at the site of the emerging
primitive streak [6]. In this way the proximo-distal signal-
ing anticipates the anterior-posterior patterning [6,7]. Pat-
terning thus occurs through a combination of tissue
interactions and cell movements [reviewed [8]].
The stages of mouse development between implantation
and the gastrulating egg cylinder have been relatively little
studied. This is due partly to the relative inaccessibility of
embryos within the uterine deciduae during this time, and
partly to their relatively poor development in culture com-
pared with preimplantation and gastrula stages. More
recently, much attention has been focused on the events
preceding gastrulation and their relation to earlier preim-
plantation development, providing an incentive to iden-
tify novel genes with restricted expression patterns during
these stages.
Several recent microarray screens have focused on stage-
specific expression in pre-implantation embryos [9-11],
whilst other screening strategies have targeted specific tis-
sues of post-implantation embryos [12-15]. In an effort to
identify new genes that are differentially expressed along
the proximo-distal axis and may have roles in early pre-
gastrula patterning events, we employed microarray anal-
ysis to compare gene expression between proximal and
distal halves of the E5.5 egg cylinder. The proximal half
includes extraembryonic ectoderm and the proximal por-
tion of the visceral endoderm, while, the distal half
includes the epiblast and the distal portion of the visceral
endoderm. After secondary screening by in situ hybridisa-
tion, we identified both known and novel genes with pre-
viously unreported differential expression in the early
mouse egg cylinder.
Results
We compared gene expression between the proximal and
distal halves of the E5.5 egg cylinder by microarray analy-
sis to identify genes with previously unreported differen-
tial expression at this stage of development. A scatter plot
of expression levels in proximal and distal segments
reveals a large number of genes that putatively show such
differential expression (Fig. 1). Several genes with previ-
ously reported differential expression in the egg cylinder
showed relative hybridisation levels consistent with such
expression patterns. These included Otx2 [16], Cripto [17],
Dnmt3b [18] and Oct4 [19] distally, and Gjb3 [20], Pem
[21], Igf2 [22] and H19 [22] proximally. We therefore
wished to test whether other previously uncharacterised
genes were also differentially expressed. We selected 40
genes, partly on the basis of differential and absolute lev-
els of hybridisation in the microarray and partly on their
likely involvement in developmental pathways, and fur-
ther screened these genes by in situ hybridisation. Of
these, we successfully identified 9 genes with previously
unreported differential expression at E5.5 and E6.5, while
the remainder showed either undetectable or ubiquitous
expression. The 9 genes and their expression patterns are
shown in Figures 2 and 3 and summarised in Table 1. In
situ hybridisation was extended to later stages from E7.5
to E9.5 for several genes, including Cubilin, Jarid1b,
Sfmbt2, Ndrg1, Talia and Plet1. However no tissue specific-
expression within embryonic tissues was identified for
any of these gene later than E8.5 (not shown), aside from
continued extraembryonic expression for Cubilin, Sfmbt2,
Ndrg1, and Plet1.
Expression patterns fell into several broad categories.
Peg10, Ctsz and Cubilin were expressed in the visceral
endoderm mainly within the proximal or "extraembry-
onic" part of the egg cylinder. The expression of Cubilin
also extended into the distal portion of the egg cylinder in
the form of two lateral "wings" overlying the epiblast. Tis-
sue sectioning showed that this expression corresponds to
the distal extent of cuboidal visceral endoderm cells (Fig.
3a, b).
Jarid1b (also called Plu-1 or Rb-Bp2) was expressed
strongly in the epiblast at E5.5 but more weakly and ubiq-
uitously at E6.5 (Fig. 2) and later stages (not shown). RT-
PCR suggested a higher level of expression in whole E7.5
embryos than in adult tissues, with the exception of strong
expression in the brain (Fig. 4).
Two genes, and Gjb5 and Sfmbt2, were expressed through-
out the extraembryonic ectoderm. In sectioned embryos,
Sfmbt2 expression appeared uniform within the chorionic
ectoderm and also within the ectoplacental cone until at
least E7.5 (Fig. 3c, d). Sfmbt2 expression was also detected
in all adult tissues tested but was substantially stronger in
brain, lung and spleen compared with heart, kidney and
liver (Fig. 4).
Ndrg1 was expressed uniformly throughout the extraem-
bryonic ectoderm but more strongly within the labyrinth
of the ectoplacental cone in all stages examined (Fig. 2,
Fig. 3). At E7.5 and E8.0 (Fig. 2, 3f), expression was also
present in the node, while becoming weaker within the
chorionic ectoderm. No specific expression in other
embryonic tissues was detected later at either E8.5 or E9.5
(not shown).
Plet1 was also specifically expressed in the extraembryonic
ectoderm, but restricted to its distal-most part as early as

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E5.5 as well as a separate domain of much stronger expres-
sion within the ectoplacental cone. The distally-restricted
expression persisted, but becoming weaker and restricted
to the peripheral chorion, until at least E8.5 (Fig. 2, 3g–h).
Expression was also detected within the ventral layer of
the node (Fig. 3i).
Talia
A previously undescribed gene corresponding to clone
H3001D07-3 was also uniformly expressed in the extrae-
mbryonic ectoderm at E5.5, and by E6.5 was also
restricted to its more distal part, adjacent to the epiblast.
Although levels appeared lower at later stages, expression
appeared to be strongest around the perimeter of the dis-
tal part of the chorionic ectoderm at E7.5 (Fig. 3j). Expres-
sion was detected ubiquitously in all adult tissues
examined by RT-PCR (Fig. 4).
A BLAST search of genomic databases identified the gene
as mapping to region XA2 of the murine X-chromosome.
A human orthologue was also identified in the syntenic
region Xq24 of the human X-chromosome and in the
marsupial Monodelphis domestica by sequence database
searches, indicating that the gene is conserved in mam-
mals. The 5'-most part of the predicted transcript also
showed homology to another previously described
human gene, XE7, which maps to Xp22.3 of the X-chro-
mosome and was originally identified as a pseudoauto-
somal gene that escapes X inactivation [23,24] and
encodes a cell surface glycoprotein expressed in trophob-
last and lymphocytes [25]. The murine gene represented
by clone H3001D07-3 we thus named Talia (a Polish
word for "waistline") to reflect its belt-like expression pat-
tern in the distal part of the extraembryonic ectoderm.
Sequence analysis of Talia and XE7
Analysis of genomic sequence data and ESTs revealed 7
exons for murine Talia with a predicted mRNA length of
6152 nucleotides (Fig. 5, 6). Promoter prediction software
[26] indicated an additional promoter and transcription
Scatter plot of microarray data comparing relative levels of gene expression for proximal and distal parts of the E5.5 egg cylin-der (x – distal, y – proximal)
Figure 1
Scatter plot of microarray data comparing relative levels of gene expression for proximal and distal parts of the E5.5 egg cylin-
der (x – distal, y – proximal). Points representing several previously published genes (Oct4, Otx2, Cripto) that display clear differ-
ential expression between proximal and distal parts are circled.

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initiation site within the 5' part of exon 7, suggesting alter-
native primary transcripts. However probes specific for
sequences either 5' or 3' of this position showed indistin-
guishable expression patterns by in situ hybridisation (not
shown), suggesting that either the latter putative promoter
is non-functional or that both are functional but share
common regulatory mechanisms.
Talia and XE7 showed homology between exons 3–5 of
murine Talia and exons 2–4 of human XE7 (Fig. 6, 7).
BLAST searches revealed numerous ESTs derived from var-
ious tissue sources that were concluded to represent
murine Xe7, despite its apparent absence from current
genomic databases. BLAST searches also identified ESTs
representing genes with homology to this conserved
Whole-mount in situ hybridisation of genes identified in microarray screen with differential expression patterns at stages as indicated
Figure 2
Whole-mount in situ hybridisation of genes identified in microarray screen with differential expression patterns at stages as
indicated. The two scale bars represent respectively 100 µm for all E5.5 and E6.5 images and 200 µm for all E7.5 and E8.0
images.

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Longitudinal sections of embryos after whole-mount in situ hybridisation, showing expression of: Cubilin at E6.5 (a) and E7.5 (b)
Figure 3
Longitudinal sections of embryos after whole-mount in situ hybridisation, showing expression of: Cubilin at E6.5 (a) and E7.5 (b);
Sfmbt2 at E6.5 (c) and E7.5 (d) (note that due to the distorted shape of the specimen this section does not fully pass through
the lumen of the proamniotic cavity and the distal-most group of Sfmbt2-expressing cells in fact forms part of the extraembry-
onic ectoderm near the anterior amniotic fold); Ndrg1 at E6.5 (e) and E7.5 (f); Plet1 at E6.5 (g), E7.5 (h) (note also that the dis-
tal-most extraembryonic expression represents extraembryonic ectoderm near the anterior amniotic fold) and in the node at
E8.0 (i); and Talia at E7.5 (j).
Table 1: Summary of genes with restricted expression patterns
NIA clone ID Gene nameTissue with specific expression
H3001E07-3
H3017E04-3
H3004E08-3
H3041C04-3
H3031C12-3
H3001A06-3
H3016G11-3
H3001D07-3
H3011D11-3
Peg10
Ctsz
Cubilin
Jarid1b
Ndrg1
Sfmbt2
Gjb5
Talia
Plet1
proximal VE
proximal VE
proximal VE
epiblast + ectoplacental cone
extraembryonic ectoderm + node
extraembryonic ectoderm
extraembryonic ectoderm
extraembryonic ectoderm
extraembryonic ectoderm + node