Dispatch R401

Mammalian development: New trick for an old dogAnthony Graham

A recent study has shown that the T-box geneeomesodermin, which was first identified in Xenopus,not only plays a conserved role in vertebrates directinggastrulation, but interestingly in mammals has acquireda new function in the development of the trophoblast.

Address: Molecular Neurobiology Group, 4th Floor New Hunts House,Guys Campus, Kings College London, London SE1 9RT, UK.E-mail: anthony.graham@kcl.ac.uk

Current Biology 2000, 10:R401–R403

0960-9822/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.

Mammals are viviparous, their embryos developing andbeing nurtured within the safe uterine environment. Thisreproductive strategy has been very successful, as it greatlyincreases the chances of survival of the neonate, and it hasinvolved major adaptations of early embryogenesis. In con-trast to other vertebrate classes, the early phase of mam-malian development is not concerned with establishingand organising the embryonic body plan, but rather time isspent generating the various extra-embryonic tissues whichare essential for the embryo to implant into the uterinewall [1]. Obviously, if one is to understand how the earlyevents in mammalian development are controlled, onemust examine mammalian embryos, but, as with manyother aspects of developmental biology, insights can comefrom unexpected places. Eomesodermin, a T-box gene firstcharacterised in Xenopus as being a key regulator of meso-derm formation [2], has recently been functionallyanalysed in mice [3]. This work has shown that, besideplaying a conserved role in mesoderm formation, this genehas also acquired a novel function in regulating one of theearliest events in mammalian development — the genera-tion of the trophoblast lineage.

Eomesodermin was first isolated in Xenopus as part of ascreen for clones that were specifically expressed in thegastrula stage embryo [2]. An interesting feature of thisclone, from amongst the 120 that were initially isolated,was that it was expressed in the equatorial region of theearly gastrula. In fact, further expression analysis demon-strated that this gene is expressed in all mesoderm cells,which in Xenopus lie as a band at the equator of the earlyembryo, and it was from this characteristic that the nameof this gene arises — from the Greek Eoσ, for dawn, indi-cating its early expression. The sequence of this clonerevealed that the gene encodes a member of the T-boxfamily of transcription factors [4], which are defined by ashared DNA binding domain, and functional analysis hassuggested that this gene plays a key role in regulating

Xenopus mesoderm differentiation [2]. If eomesodermin isectopically expressed in animal caps, then it induces thetranscription of early mesodermal genes. Moreover, Eome-sodermin acts in a concentration-dependent manner, withhigher levels inducing more dorsal cell types. Correspond-ingly, if eomesodermin function is interfered with, thenembryonic development is arrested during gastrulation,and mesoderm formation is severely perturbed.

The mouse eomesodermin gene shares many characteristicswith its Xenopus orthologue, displaying a high degree ofsequence relatedness — up to 95% amino acid sequenceidentity in the T-box region [3,5] — as well as prominentexpression in the mesodermal cells of the gastrula stageembryo. The mouse eomesodermin gene is notable,however, as it is also expressed at the very early blastocyststages of development [3,6,7]. The blastocyst consists oftwo groups of cells, the internally located inner cell massand the externally located trophectoderm (Figure 1), andit is this latter group of cells which first expresses eomeso-dermin. The specification of trophectoderm versus theinner cell mass is the earliest cell-fate choice in mam-malian development, and the one which separates thecells of the trophoblast lineage, which form the foetalportion of the placenta, from the embryonic lineages.

The function of eomesodermin in this peculiarly mammalianphase of development has recently been uncovered bymutational analysis in mice [3]. While heterozygousanimals, which carry a normal and a mutated form of eome-sodermin, are healthy and fertile, they never yield anyhomozygous mutant offspring when crossed, suggestingthat eomesodermin function is absolutely required fornormal development. Morphological analysis of mutantembryos showed that, in the absence of eomesodermin,embryos arrest soon after implantation.

Eomesodermin mutant embryos reach the blastocyst stage,generating trophectoderm and the inner cell mass, but thetrophectodermal cells are unable to progress and form thetrophoblast [3]. If normal blastocyst embryos are grownin vitro, they hatch from the zona pellucida, which hasprotected them since fertilisation, and settle, andtrophoblast cells readily form and spread across theculture dish. But although cultured mutant embryos hatchnormally, they maintain their blastocyst morphology andtrophoblast cells never form. It has been shown that tro-phoblast stem cell lines can be derived from blastocysts inculture, that these cells strongly express eomesodermin andthat their maintenance requires the presence of thefibroblast growth factor FGF-4 [8]. The lack of production

of trophoblast cells from eomesodermin mutant embryoscannot be overcome by the addition of FGF-4, however,and it seems that eomesodermin must be required cellautonomously for the initial transition from trophecto-derm to trophoblast [3].

While the fact that eomesodermin is required for thedevelopment of the trophoblast is in itself of great inter-est, the early lethality that results in the absence of thisgene means that one cannot quite so straightforwardlyanalyse its later roles in development. Of particular inter-est would be to probe the role of this gene in gastrulationin the mouse, as it is prominently expressed in the cells ofthe primitive streak and mesodermal cells that haveemerged from there (Figure 1). However, mouse embryol-ogists have devised the trickery necessary to circumventthis problem.

One can create chimeras in which the extra-embryonictissues are wild-type, but the embryonic tissues are mutant,by introducing mutant embryonic stem cells, or inner cellmasses, into a tetraploid host; tetraploid cells can only gen-erate extra-embryonic tissues. When this is done, thechimeras show no obvious abnormalities at early gastrula-tion stages, indicating that the wild-type cells have formedthe trophoblast [3]. At slightly later stages, however, defectsbecome apparent. Although the primitive streak forms, itdoes not elongate and the node, the organising center of themammalian embryo, is absent from its anterior end [3]. Thefact that the primitive streak forms demonstrates that thenecessary early mesoderm inducing signals are present, butit seems that the mesoderm never emerges from the primi-tive streak. Analysis of the prospective neural tissue of themutants also reveals that the anterior patterning of thisembryonic layer is slightly perturbed [3].

This results mirrors what was observed in Xenopus,suggesting that eomesodermin has a conserved role ingastrulation and that the absence of its function results ingastrulation arrest. This phenotype could, however be dueto this gene being necessary for mesoderm differentiationor, more specifically, for the recruitment of prospectivemesodermal cells into the primitive streak, or indeed both.While the experiments in Xenopus do not allow us to differ-entiate between these two scenarios, the analytical toolsavailable in mice do. In chimeras made from diploid wild-type cells and mutant cells, mesoderm formation is partiallyrescued [3]. In this situation, wild-type and mutant cellsintermingle in the epiblast, but only the wild-type cellsmigrate through the primitive streak; the eomesoderminmutant cells remain within the epiblast. In normal develop-ment, the differentiation of cells into mesodermal tissuesfollows from their migration through the primitive streak,and so the block in early gastrulation observed in the eome-sodermin mutants does not tell us as whether or not thisgene is also necessary for the differentiation of mesodermalcell types. But this issue can be addressed by producingteratomas from the mutant cells, and analysing the range ofdifferentiated cell types that are produced. When this isdone, the eomesodermin mutant cells form muscle, cartilageand haemopoetic tissue. It thus seems that the function ofeomesodermin during gastrulation is in directing the migra-tion of cells from the epiblast into the primitive streak.

The results from the mutational analysis of the function ofeomesodermin in mice [3] are interesting for a number ofreasons. They confirm that this gene has a prominent rolein controlling gastrulation across the vertebrates. Theresults also, however, demonstrate that eomesodermin hasacquired a novel role in mammals, being now alsoinvolved in controlling one of the very earliest develop-mental events — the generation of the trophoblast. Yet, atthe cellular level, eomesodermin may be playing a similarrole in both trophoblast and mesodermal cells. Both of

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Figure 1

(a) Schematic diagram of a blastocyst — a 3.5 day mouse embryo. Thelocations of the inner cell mass and the trophectoderm are highlighted.(b) Schematic diagram of a 6.5 day mouse embryo during the earlyperiod of gastrulation. As gastrulation proceeds, the cells of thepresumptive mesodermal cells of epiblast move to and through theprimitive streak, shown by blue arrows. The primitive streak itself willalso extend anteriorly, indicated by the red arrow, and at its anteriorend the node will be established.

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Visceralendoderm

Trophectoderm

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these cell types exhibit pronounced migratory behaviour,and in the absence of this gene trophoblast cells do notspread and migrate in culture and mesodermal cells do notmigrate into the primitive streak.

The most likely explanation for the emergence of eomeso-dermin function in the trophoblast lineage must be that, inthe events leading to the evolution of the mammals, thisgene acquired novel regulatory elements that drove itsexpression in the trophectoderm of the blastocyst stageembryo. It would therefore be very exciting to probe thecontrol elements that drive eomesodermin expression in themouse, to determine whether one can identify trophecto-derm-specific regulatory sequences. It would also be inter-esting to compare the regulatory regions of eomesoderminfrom representatives of different vertebrate classes, to tryand identify conserved elements that possibly controlexpression of this gene during gastrulation, and further toget an insight into the evolutionary changes that allowedeomesodermin to acquire a novel function.

References1. Cruz YP: Mammals. In Embryology: Constructing the organism Edited

by Gilbert SF and Raunio AM. Sauer Associates; 1997:459-489.2. Ryan K, Garrett N, Mitchell A, Gurdon JB: Eomesodermin, a key

early gene in Xenopus mesoderm differentiation. Cell 1996,87:989-1000.

3. Russ AP, Wattler S, Colledge WH, Aparicio SAJR, Carlton MBL,Pearce JJ, Barton SC, Surani MA, Ryan K, Nehls MC, et al.:Eomesodermin is required for mouse trophoblast developmentand mesoderm formation. Nature 2000, 404:95-99.

4. Smith JC: T-box genes: what they do and how they do it. TrendsGenet 1999, 15:154-158.

5. Wattler S, Russ A, Evans M, Nehls M: A combined analysis ofgenomic and primary protein structure defines the phylogeneticrelationship of new members of the T-box family. Genomics 1998,48:24-33.

6. Ciruna BG, Rossant J: Expression of the T-box gene Eomesoderminduring early mouse development. Mech Dev 1999, 81:199-203.

7. Hancock SN, Agulnik SI, Silver LM, Papaioannou VE: Mapping andexpression analysis of the mouse ortholog ofXenopus Eomesodermin. Mech Dev 1999, 81:205-208.

8. Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J: Promotionof trophoblast stem cell proliferation by FGF4. Science 1998,282:2072-2075.

Dispatch R403

If you found this dispatch interesting, you might also wantto read the August 1999 issue of

Current Opinion inGenetics & Developmentwhich included the following reviews, editedby Norbert Perrimon and Claudio Stern, onPattern formation and developmentalmechanisms:

Cell polarity in the early Caenorhabditis elegans embryoBruce Bowerman and Christopher A Shelton

The polarisation of the anterior–posterior anddorsal–ventral axes during Drosophila oogenesisFredericus van Eeden and Daniel St Johnston

Wnt signaling and dorso-ventral axis specificationin vertebratesSergei Y Sokol

Establishment of anterior–posterior polarity inavian embryosRosemary F Bachvarova

Polarity in early mammalian developmentRichard L Gardner

Diverse initiation in a conserved left–right pathway?H Joseph Yost

Extracellular modulation of the Hedgehog, Wnt and TGF-βsignalling pathways during embryonic developmentJavier Capdevila and Juan Carlos Izpisúa Belmonte

Fringe, Notch, and making developmental boundariesKenneth D Irvine

Polarity determination in the Drosophila eyeHelen Strutt and David Strutt

Wnt signalling: pathway or network?Alfonso Martinez Arias, Anthony MC Brown and Keith Brennan

Epithelial cell movements and interactions in limb,neural crest and vasculatureCheryll Tickle and Muriel Altabef

Cell movements in the sea urchin embryoCharles A Ettensohn

Roles of the JNK signaling pathway in DrosophilamorphogenesisStéphane Noselli and François Agnès

Cell migration in DrosophilaAlexandria Forbes and Ruth Lehmann

Cell migration and axon growth cone guidance inCaenorhabditis elegansCatherine S Branda and Michael J Stern

The full text of Current Opinion in Genetics & Developmentis in the BioMedNet library athttp://BioMedNet.com/cbiology/gen