Pluripotent cells (stem cells) and their determination and ...€¦ · Stem cells in vertebrate...

Develop. Growth Differ. (2001) 43, 469–502

Introduction

Two topics in developmental biology have recentlyattracted international public interest: (i) the isolationand culture of human stem cells (Shamblott et al. 1998;Thomson et al. 1998) for potential use in transplanta-tion medicine; and (ii) the cloning of a sheep nearlygenetically identical with its parent by incorporating anucleus from an adult cell into an enucleated oocyte(Wilmut et al. 1997). Obviously, the nucleus from the

adult cell was reprogrammed by the cytoplasm of theenucleated fertilized oocyte.

The scientific foundations of these recent develop-ments have a long history. The role of the cell cytoplasmand the nucleus and their cooperation in animal devel-opment is a fundamental question in biology. Early cellisolation experiments on sea urchin embryos identifiedorgan-determining substances in the egg cytoplasm(Boveri 1901). The sea urchin Paracentrotus lividuscontains a pigmented ring only in the vegetal part ofthe egg. This pigment is not involved in cell deter-mination, but allows identification of animal and vege-tal cells. Boveri found that only those cell fragmentscontaining vegetal cytoplasm could develop to the pluteus stage. The genes are located in the cell nucleusand cytoplasmic factors are necessary to determinewhich genes are activated and/or repressed.

*Author to whom all correspondence should be addressed, atAm Heselsberg 33, D-88416 Ochsenhausen, Germany.

Email: [email protected]†This article is dedicated to the memory of Dr Hildegard

Tiedemann.Received 15 April 2001; revised 16 May 2001; accepted 7 June

2001.

Review

Pluripotent cells (stem cells) and their determination and differentiation in early vertebrate embryogenesis†

H. Tiedemann,1* M. Asashima,2 H. Grunz3 and W. Knöchel4

1Institut für Molekularbiologie und Biochemie der Freien Universtität Berlin, Arnimallee 22, D-14195Berlin, 3Universität Essen, Abteilung für Zoophysiologie, D-45117 Essen, 4Abteilung Biochemie,Universität Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany and 2Department of Life Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.

Mammalian embryonic stem cells can be obtained from the inner cell mass of blastocysts or from primordialgerm cells. These stem cells are pluripotent and can develop into all three germ cell layers of the embryo.Somatic mammalian stem cells, derived from adult or fetal tissues, are more restricted in their developmentalpotency. Amphibian ectodermal and endodermal cells lose their pluripotency at the early gastrula stage. Thedorsal mesoderm of the marginal zone is determined before the mid-blastula transition by factors located aftercortical rotation in the marginal zone, without induction by the endoderm. Secreted maternal factors (BMP, FGFand activins), maternal receptors and maternal nuclear factors (�-catenin, Smad and Fast proteins), which formmultiprotein transcriptional complexes, act together to initiate pattern formation. Following mid-blastula transi-tion in Xenopus laevis (Daudin) embryos, secreted nodal-related (Xnr) factors become important for endodermand mesoderm differentiation to maintain and enhance mesoderm induction. Endoderm can be induced byhigh concentrations of activin (vegetalizing factor) or nodal-related factors, especially Xnr5 and Xnr6, whichdepend on Wnt/�-catenin signaling and on VegT, a vegetal maternal transcription factor. Together, these andother factors regulate the equilibrium between endoderm and mesoderm development. Many genes are activated and/or repressed by more than one signaling pathway and by regulatory loops to refine the tuningof gene expression. The nodal related factors, BMP, activins and Vg1 belong to the TGF-� superfamily. Thehomeogenetic neural induction by the neural plate probably reinforces neural induction and differentiation.Medical and ethical problems of future stem cell therapy are briefly discussed.

Key words: early differentiation, ethical problem, human stem cell, stem cell, vertebrate embryo.

470 H. Tiedemann et al.

Pluripotent cells in amphibian embryos andtest methods for inducers

The cleaving amphibian egg first forms a morula, fol-lowed by the blastula stage, when a cavity called theblastocoele develops. At the gastrula stage, a blasto-pore is formed in the presumptive endoderm. It isthrough the blastopore that the endoderm, the mesen-doderm on the leading edge of the mesoderm andmost of the mesoderm (the so-called marginal zonebetween the animal ectoderm and the vegetal endo-derm; Fig. 1) invaginates. The invaginated dorsalmesoderm induces development of the neural plate inthe overlying ectoderm. This forms the primordium ofthe neural anlage. If small pieces of tissue from theectoderm and the neural anlage are exchanged in theearly gastrula stage, the grafted tissue develops in con-formity with its new surroundings. The grafted ectodermbecomes part of the neural plate and the grafted pre-sumptive neural plate becomes ectoderm (Spemann1918). These experiments indicate that the grafted tissues from the amphibian gastrula are still pluripotent.

The ectoderm of early gastrulae has been shown toform beside neural tissues somites when the pre-sumptive mesoderm of the dorsal blastoporal lip (theorganizer) is transplanted into the ventral ectoderm ofa host gastrula (Spemann & Mangold 1924). The neuraltube of the induced secondary axis system wasderived from the host ectoderm and the chimericsomites were derived from the host ectoderm and thetransplanted blastoporal lip. Holtfreter (1933a,b, 1939)then applied a sandwich technique (tissue fragmentsor pellets that contain inducing factors are envelopedby two pieces of explanted ectoderm from early gas-trulae) to show that ectoderm can be induced by anumber of other tissues. The multipotency of the ecto-derm has also been used in implantation techniques(‘Einsteckmethode’; Mangold 1923) to test inducingmaterial. In these experiments, tissue fragments or pellets containing inducing factors (highly active fac-tors can be mixed in different proportions with a non-inducing protein, such as �-globulin) are implantedthrough a slit into the blastocoele of an early gastrula.The gastrulation movements cause the pellet to comeinto contact with the pluripotent ventral ectoderm.However, this method is less suitable for Xenopus laevis embryos, because a splitting of the axis canoccur. Substances in solution can be tested on isolatedectoderm from blastulae or early gastrulae (animal captest; Becker et al. 1959). In another test, mRNA for theprotein to be tested is injected into blastomeres, thenthe animal cap is excised at the blastula stage andtested for tissue-specific markers. The formation ofaxes after injection of RNA into blastomeres (of normal

or UV-irradiated eggs; Scharf & Gerhart 1980) is a further, more complex test method.

Methods to test induction of tissue developmentdepend on the pluripotency of the ectoderm (animalcap). The ectoderm is, however, not completely ‘undif-ferentiated’. Ectoderm that is isolated and cultured insalt solution becomes ciliated and develops to atypi-cal epidermis, with an ultrastructural differentiation similar to its development in vivo (Billet & Courtenay1973; Grunz 1973; Grunz et al. 1975). These structures,however, lack the basal lamina and the regular align-ment of epidermal cells, which depends on mesen-chyme (Holtfreter 1939).

The fate of ectoderm is reversible up to the early gas-trula stage. When labeled single animal pole blasto-meres from X. laevis blastulae are transplanted into theblastocoele of late blastula host embryos, the daugh-ter cells of the transplanted blastomeres contribute toall three germ-cell layers, in agreement with the trans-plantation and induction experiments. At later stages,the fate of the transplanted animal cells becomesrestricted solely to ectoderm (Snape et al. 1987). Singlevegetal pole blastomeres from X. laevis morulae that

Fig. 1. Map of the presumptive regions of the blastula of theurodele, Cynops pyrrhogaster, projected onto the surface of theembryo as seen from the left side. In the animal half higher thanthe limit of invagination (Eg) are the (E) epidermis, (Gl) neuralcrest, (N) neural plate, (T) tail somites and (t) lateral mesodermin the tail. In the marginal zone are the (Ch) notochord, (1–9) numbering of trunk somites, (P) posterior trunk somites, (PrC) pre-chordal mesoderm and (Spl) lateral plate mesoderm in the trunk.In the vegetal part are the (D) endoderm of archenteron, (J) start-ing point of invagination, (U) boundary between the mesodermand endoderm (prospective lateral lip of the blastopore at thebeginning). After Nakamura (1942) from Annot. Zool. Jap. 21,161.

Stem cells in vertebrate embryogenesis 471

are transplanted into the blastocoele of host embryoscan contribute progeny to all cell layers. By the earlygastrula stage, the cells contribute only to the endo-derm. This process is seen in vitro when an appropri-ate tissue mass is present (Heasman et al. 1984; Wylieet al. 1987). The differential expression of cell surfacemolecules to change cell affinities is an important factor for these experiments.

The marginal zone (presumptive mesoderm) is deter-mined to mesoderm early in the development process.Isolated marginal zones from X. laevis and Trituruspyrrhogaster have the capacity for self-differentiationfrom the early blastula stage onwards (Nakamura &Tarasaki 1970; Nakamura et al. 1970; Nakamura &Toivonen 1978), which shows that mesoderm is deter-mined before the mid-blastula transition.

The attempts to characterize induction factors havebeen confused by inadequate methodology. This ledto the still-existing prejudice that tests on Triturusalpestris, T. pyrrhogaster, Pleurodeles waltl and othersled to neuralization without inducer, the so-called‘autoneuralization’. However, autoneuralization wasonly observed in ectoderm explants from Ambystomamexicanum early gastrulae in Flickinger solution as the culture medium (Holtfreter 1944; Holtfreter &Hamburger 1955; Tiedemann 1986b).

Mammalian pluripotent cells (embryonic stemcells) from blastocysts and primordial germcells

In mammalian development, the cells of the inner cellmass of the blastocyst, the mother cells of the embryoproper, are multipotent for a short period. When thesecells are transplanted to ectopic sites, for example innude mice, they form teratocarcinomas. Invasivegrowth, which is characteristic for carcinomas, was alsoobserved in a low percentage when the ectoderm ofearly T. alpestris gastrulae was implanted into the visceral cavity between the somatopleura and liver ofadult Triturus taeniatus (Waechter 1951). Martin (1981)and Evans and Kaufman (1981) were able to maintaincells from the inner cell mass of mice in tissue culture,where they could be continuously propagated aspluripotent embryonic stem (ES) cells. If injected backinto the blastocyst of a host female, the cultured stemcells can contribute to many tissues of the chimeric off-spring. Subcutaneous injection of ES cells into syn-geneic mice induces teratomas, which contain cells ofectodermal, mesodermal and endodermal origin(Wobus et al. 1984). Pluripotent stem cells from othermammalian species have also been maintained in tissue culture (Shamblott et al. 1998 and referencestherein). Mouse and other mammalian stem cells are

characterized by expression of the germ-line tran-scription factor Oct 4 (Nichols et al. 1998), high levelsof alkaline phosphatase and the expression of certaincell surface glycolipids (Solter & Knowles 1978) andglycoproteins (Andrews et al. 1984). These proteins arecharacteristic, but not specific, for stem cells.

Stem cells are mostly cultured in medium supple-mented with 10–20% fetal bovine serum or horseserum, which contains small amounts of growth and differentiation factors. Leukemia inhibitory factor (LIF;Smith 1991) is added to the culture medium to preventdifferentiation, and recombinant basic fibroblast growthfactor (bFGF) is added to enhance multiplication of thestem cells. Culture in serum-free defined media is pos-sible, but the replication of cells is retarded comparedwith cells cultured in media with serum.

On withdrawal of LIF, the cells aggregate to formembryoid bodies, which can be cultured under differ-ent conditions (for review see Keller 1995). Within thefirst few days of differentiation, the embryoid bodiesexpress marker genes of primitive endoderm andmesoderm. After extended periods in culture, tissuesof all three germ layers develop.

The induction and development of hematopoietic(see also later), muscle and neural cell lineages hasbeen well studied. Activin and bone morphogeneticprotein (BMP-4; see later) induce mesoderm formationand hematopoiesis in embryoid bodies cultured inserum-free defined medium (Johansson & Wiles 1995).A blastocyst-derived ES cell line expresses muscle-specific genes in embryoid bodies in a temporal pat-tern that precisely reflects the sequence in vivo in themouse (Rohwedel et al. 1994). An enriched prepara-tion of cardiomyocytes has been isolated by micro-dissection of the cardiogenic regions of embryoidbodies. In these cells, DNA synthesis is markedlydecreased 21 days from induction, with a concomitantincrease in multinucleated cells (Klug et al. 1995). Toenrich the cardiomyocytes, multipotent ES cells weregenetically modified to express both a gene constructcarrying an �-cardiac myosin heavy chain (MHC) promoter–aminoglycoside phosphotransferase and aphosphoglycerate kinase–hygromycin resistance trans-gene. Transfected cardiomyocytes were selected bygrowth in the presence of hygromycin, then trans-planted successfully into the hearts of adult dystrophicmice. Loss of cardiomyocytes in the mammalian heartis irreversible and can lead to diminished cardiac func-tion; thus, cardiomyocyte transplantation has significanttherapeutic potential (Klug et al. 1996).

Neuronal precursor cells and functional post-mitoticneurons have been obtained from ES cells in vitro(Okabe et al. 1996). Aggregates of mouse ES cellstreated for 4 days with retinoic acid showed a high rate


of neuritic outgrowth. When these cells were replatedas a monolayer, many cells had a neuronal appearanceand expressed neuronal markers. The cells generatedaction potentials and expressed tetrodotoxin-sensitivesodium channels (Bain et al. 1995). Cells expressingglial precursor markers were obtained from aggregatesof mouse ES cells by propagating them sequentially ina medium containing bFGF, epidermal growth factor(EGF) and finally both bFGF and platelet-derivedgrowth factor (PDGF). On growth factor withdrawal, thecells differentiated in either oligodendrocytes or astro-cytes (Brüstle et al. 1999).

Transplantation experiments have shown that neuralprecursor cells generated in vitro can participate in thedevelopment of embryonic rat brain (Brüstle et al.1997). Following transplantation of glial precursor cellsinto the ventricle of myelin-deficient rat brains, myelinsheets formed in various brain regions. The ethical con-siderations involved in the therapeutic application ofthese type of experiments to human beings have alsobeen discussed (Brüstle & Wiestler 2000). A humanstem-cell lineage equivalent to the mouse glial cell line may be used to treat diseases such as multiplesclerosis, which primarily affects glial cells. Systematictrials with combinations of differentiation-inducing factors to attain a special lineage pathway of stem cellswill in the future be a difficult, but rewarding, task.

Human ES cells have been derived from human blastocysts. The cultured stem cells differentiate to formall three embryonic germ layers when grown to con-fluence. Differentiation can be induced even after several months of culture as undifferentiated cells(Thomson et al. 1998). In vitro aggregation of humanstem cells induces embryoid bodies (as with othermammalian stem cells). In addition to other differenti-ated tissues derived from all three germ layers, a minority of embryoid bodies show rhythmic pulsing ofprecursor myocardial cells expressing �-cardiac actin(Itskovitz-Eldor et al. 2000).

Pluripotent stem cells can also be obtained from primordial germ cells (Matsui et al. 1991; Resnick et al.1992). A mouse embryonic germ cell line has been cul-tured as embryo-like aggregates. The differentiationefficiency of this line into cardial and muscle cells waslower and the spontaneous neuronal differentiationhigher as compared with an ES cell line (Rohwedel et al. 1996). To establish pluripotent stem cell lines fromhuman primordial germ cells, gonadal ridges andmesenteries of 5–9-week-old post-fertilization humanembryos (obtained as a result of therapeutic termina-tion of pregnancy) containing primordial germ cellshave been cultured on feeder layers after disaggre-gation (Shamblott et al. 1998). Human recombinantbFGF (see later), human LIF and forskolin were added

to the culture medium. Colonies of primordial germ cellswere identified by alkaline phosphatase and immuno-logic markers routinely used to characterize ES cellsand embryonic germ cells. As with ES cells, pluripo-tent embryonic germ cells can aggregate to formembryoid bodies, indicating that cell–cell interaction isessential for cell differentiation. Embryoid bodies areoften surrounded by a layer of endoderm and containa mixture of differentiated cells; however, the restrictedgrowth characteristics of differentiated cells in embry-oid bodies limits their use. Human embryoid bodiesgrown in the presence of LIF, FGF and fetal calf serumhave been enzymatically dissociated to derive clonalcell lines. The embryoid body-derived (EBD) cells showrobust and long-term proliferation with a normal karyo-type and the ability to be cryopreserved. They can bestably transduced with either lentiviruses or retrovirusesand preferentially express neural markers. They alsoexpress endodermal, vascular, hematopoietic andmuscle cell markers (Shamblott et al. 2001).

Parkinson’s disease arises from a loss of dopaminenerve terminals in the striatum. Recently, precursors ofdopaminergic neurons in cultured mesencephalic cellsobtained from human embryos, aborted 7–8 weeksafter conception, were transplanted to Parkinson’s disease patients. Some of the implanted cells survivedand differentiated (18F-fluorodopa scanning by positronemission tomography (PET)) in the absence of immuno-suppressive therapy. A small improvement wasobserved during the first year after surgery. Thereafter,severe spontaneous dyskinesias developed as a dis-abling complication in five patients (15%; Greene et al.1999; Freed et al. 2001). This trial highlighted the needto know more about the molecules synthesized by theneurons, and the proliferation, migration, differentiationand survival of the precursor cells after transplantation(Fischbach & McKhann 2001).

Experiments to elucidate the molecular mechanismof ES cell propagation in a pluripotent state have iden-tified the importance of the transcription factor STAT3,which is activated by a LIF–glycoprotein (gp) 130receptor complex. STAT3 has four docking sites ongp130, which are not functionally equivalent. The transcription factor is, however, also involved in the dif-ferentiation of somatic cells. Cell type-specific inter-pretation of STAT3 activation may explain its diversedevelopmental effects (Niwa et al. 1998).

Stem cells in somatic mammalian tissues and organs

Soon after the blastocyst stage, pluripotent stem cellsgive rise to primitive germ-line stem cells (see earlier)and somatic progenitor cells, which can only be dealt


with briefly in this review. Hematopoietic stem cells arethe most thoroughly investigated stem cell system (forreview see Duesenbery 1998; Fuchs & Segre 2000;Weissman 2000 and references therein). Hematopoieticstem cells in the blood islands of the yolk sac providehematopoiesis. From the embryonic loci, hemato-poietic stem cells migrate to the fetal liver and fromthere to the spleen and the bone marrow. Long-termhematopoietic stem cells (Weissman 2000 and refer-ences therein) probably have a high turnover.Approximately 8–10% of these cells randomly enter thecell cycle per day in young adult mice. In man andmouse, chemotherapy (acute myelosuppression;Richman et al. 1976) and treatment with the cytokinegranulocyte colony stimulating factor, a glycoprotein,initiate mitosis and mobilize stem cells to migrate intothe blood stream (Metcalf et al. 1980; Durhsen et al.1988; Molineux et al. 1990a,b). Blood from mice treatedwith granulocyte colony stimulating factor can rescuelethally irradiated mice (Molineux et al. 1990b), as seenwith bone marrow transplantation.

Cell-sorter based separation of bone marrow cellslabeled by monoclonal antibody or stained with fluor-escent dyes led to the isolation of hematopoietic stemcells in the mouse (Spangrude et al. 1988; Goodell et al. 1996). The pluripotent stem cells pass throughstages of progenitors, which are themselves still stemcells, but are more restricted in their options. Each stepof stem-cell differentiation involves further maturation.At the end of this process, common lymphocyte pro-genitors arise, which give rise to T lymphocytes, B lymphocytes and killer cells, as well as progenitors ofthe myeloerythroid lineages. The latter give rise tomyelomonocytic and megakaryotic/erythroid precur-sors. Combinatorial interactions of signal moleculesand positive and negative transcription factors controlthe advancing differentiation, as in early embryos (seelater). Renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated bystromal cells and serum factors (Spooncer et al. 1986).The G1-protein-coupled CXC chemokine receptor 4 isactivated by stromal cell-derived factor 1 (SDF-1). The expression pattern of chemokine receptor 4 of X. laevis suggests that SDF-1 and the CXCR4 recep-tor are involved in regulating the migratory behavior ofhematopoietic stem cells colonizing the larval or fetalliver (Moepps et al. 2000).

The glycoprotein erythropoietin, which is producedprimarily in the adult kidney, plays an important role inthe regulation of erythropoiesis (Goldwasser 1967). Inembryos lacking a functional erythropoietin receptor in the yolk sac, due to targeted disruption of the receptor gene, primitive erythrocytes are produced in normal numbers, although they are reduced in size

and their proliferation is retarded. In contrast, definitiveerythropoiesis in the fetal liver is drastically inhibitedbeyond the late progenitor stage. This suggests thatearly and late erythropoiesis are differentially regulated.It is possible that another cytokine and receptor playa role in early hematopoiesis (Lin et al. 1996). In pluri-potent hematopoietic stem cells, multilineage geneexpression has been observed prior to commitment toa distinct lineage (Hu et al. 1997). However, this couldto some extent depend on the culture of the cells inserum, which contains growth and differentiation fac-tors in small concentrations.

Tissues other than blood cells in the adult canundergo self renewal from somatic stem cells (reviewedby Fuchs & Segre 2000 and references therein). Thesestem cells often rapidly proliferate initially and thenundergo terminal differentiation. In contrast to ES cellsfrom the inner cell mass, the developmental potencyof these stem cells is limited. The epidermis maintainsa basal layer of dividing cells, which then withdraw fromthe cell cycle and migrate outwards to the skin surface.Cultured epidermal cells are used in medicine toenhance wound healing. The characteristic structuralelements of the small intestinal mucosa are ciliated villi.Between the villi, crypts protrude to the submucosa.Stem cells are located near or at the base of the crypts.Crypt cells express Tcf4, a member of the Lef/Tcf family of transcription factors in the Wnt/�-catenin path-way (see below). Tcf4 null mice have no stem cells inthe intestinal crypts (Korinek et al. 1999). Cultured keratinocyte stem cells of the skin have elevated levels of activated �-catenin. Signaling via �-1 integrinsand mitogen-activated protein kinase (MAPK) deter-mine human epidermal stem cell fate (Zhu et al. 1999).

Neural crest stem cells, from which all neural crestcell lines are derived, have been isolated from rats(Morrison et al. 1999). The identification of stem cellsfrom the adult central nervous system has also beenreported (Johansson et al. 1999; reviewed by Fuchs &Segre 2000; Weissman 2000 and references therein).Stem cells from adult brain have also been obtainedfrom bodies (Palmer et al. 2001).

Bone undergoes continuous remodeling duringgrowth and adult life. The cells involved are chondro-cytes and osteoblasts of mesenchymal origin, whereasthe osteoclasts derive from bone marrow monocytes.Several BMP are thought to regulate early differentia-tion of mesenchymal cells to chondrogenic andosteogenic cell lineages. The expression of BMP-4 (amember of the TGF-� superfamily, see later) isincreased at bone fracture. Human BMP-2 and BMP-7 are used, together with biomaterials (for example special ceramics), to adhere newly formed bone indelayed fracture healing, pseudoarthrosis and other


bone diseases (reviewed by Wozney 1994; Erlebacheret al. 1995; Küswetter & Teschner 1999 and referencestherein).

In addition to their potential in transplantation medi-cine, stem cells could be useful as a gene ferry for genetherapy. Genes transferred into stem cells andexpressed in the differentiated cells originating from thestem cells would be amplified together with the stemcells. One major problem with this application is thesearch for suitable vectors for the stable transductionof large genes.

Transdifferentiation

It has been known for a long time that the different tissues of the newt eye can be transformed, a processcalled transdifferentiation (Lopashov 1977). For exam-ple, retina can be spontaneously regenerated from pig-mented epithelium (Stone 1950). Retina from tadpolescan transform pigmented epithelium into retina with allthe specific cell elements, but without normal organ-ization, when the Bruch’s membrane (part of the mesenchymal development of the eye) is removed(Lopashov & Sologub 1972). This process is accom-panied by shedding of part of the cytoplasm, includ-ing pigment granules, as seen with lens regenerationfrom the iris (Yamada & McDevitt 1974). Lens tissue canalso be formed in cultures of cell lines originally derivedfrom retinal pigmented cells (Eguchi & Okada 1973).Recently, the transdifferentiation of bone marrow orblood cells to donor-derived endothelial cells (angio-genesis) and somitic muscle has been reported, as wellthe implication of bone marrow cells in liver regenera-tion (Weissman 2000 and references therein).

Neural induction and differentiation

Isolation of neuralizing factors

Factors that induce the development of neural tissuehave been partially purified from chicken brain(Mikhailov & Gorgolyuk 1989) and X. laevis gastrulastages (Janeczek et al. 1992; Tiedemann & Tiedemann1999). Exclusively neural tissues of the forehead typeconsisting of forebrain (telencepalon and diencepalon),eyes with lenses, retina, tapetum and nose are inducedby neuroblastoma cell-line homogenates (Lopashov et al. 1992).

In homogenates from X. laevis gastrula–neurulastages, neural-inducing factors are found in ribo-nucleoprotein (RNP) particles (~110 Å diameter), whichare different from ribosomal subunits, in small vesiclesand in high-speed spin supernatant (Janeczek et al.1984). These fractions and the factors isolated fromthem induce foreheads, very little hindheads and no

mesodermal tissues. The RNP particles are highlyinductive. When tested by the ‘Einstecktest’ on T. alpes-tris early gastrulae, large secondary neural platesdevelop very early. Large areas of the ventral and thelateral ectoderm are attracted by the induction, so thatthe gastrulation is disturbed. In 50% of cases, embryoswith two heads with eyes (‘Doppelköpfe’) and a rudi-mentary primary axis develop. Other embryos showlarge heads on the belly of the host larvae. The activecomponent is a basic protein in a hydrophobic basicprotein complex. The molecular weight of the maininducing protein estimated by size-exclusion high-pressure liquid chromatography (SE-HPLC) afterreduction with mercaptoethanol, which does not inac-tivate neuralizing factors, is in the range of 33–50 kDa.A neuralizing factor from the supernatant was purified800–1000-fold in the presence of protease inhibitors.After sodium dodecylsulfate (SDS)–polyacrylamide gelelectrophoresis (PAGE) separation and elution, most ofthe activity was found from 14 to 30 kDa, with lesseractivity shown at 100–110 kDa. This factor is part of anacidic protein complex, although the active protein maybe basic. After complete deglycosylation with trifluoro-methansulfonic acid, the inducing activity was not irre-versibly lost. After ultracentrifugation of the supernatant,no activity < 10 000 kDa was found. The neuralizing factor in the small vesicles resembles that found in thesupernatant. The factors could either be different pro-teins or part of a precursor-product line. The RNP par-ticles and small vesicles from unfertilized oocytes showsimilar inducing activities to the corresponding fractionsfrom gastrula stages, indicating that the factors in thesefractions are maternal factors. The supernatant fromunfertilized oocytes, however, has only a very smallneural inducing activity.

In the embryo, neural inducing factors are releasedby the endomesoderm at the leading edge of theinvaginating mesoderm and by the invaginated meso-derm (see later) to induce the dorsal ectoderm to formthe neural plate. The factor(s) are released from themesoderm in diffusible form, as shown by transfilterinduction experiments (Saxén 1961). Accordingly, asmall amount of neuralizing factor can be isolated fromthe extracellular space between the dorsal mesodermand the overlying neural plate of very early stage neu-rulae (John et al. 1983). The neural inducing activity ofthe mesoderm is in part inhibited by actinomycin D(which inhibits RNA synthesis) and by actidion (whichinhibits protein synthesis). This suggests either thatneuralizing factor(s) are synthesized de novo in themesoderm or that mRNA and protein synthesis areneeded for the release of maternal factor(s) from themesoderm (reviewed by Tiedemann et al. 1998 and references therein).


Neural inducing factors release in the dorsal ectoderm the inhibition of neuralization

Disaggregation experiments on X. laevis animal ecto-derm (animal caps) have had a strong impact on theunderstanding of neural induction. After dissociation ofthe ectoderm and dispersal of the cells for 3 h prior toreaggregation, strong neuralization occurs (Grunz &Tacke 1989). However, the neuralization is blockedwhen extracellular matrix material, which is released tothe medium after dissociation, is added before reag-gregation (Grunz & Tacke 1990; Grunz 1997, 2001).This suggests that neural inducing factors have noinstructive information for the induced ectoderm, butthat neuralization in the ectoderm is inhibited and thatthe factors from the mesoderm release this inhibition.The release of inhibition depends on direct interactionof neural inducing factors with factors like BMP-4(which is contained in the extracellular matrix in con-siderable amounts; W. Knöchel & H. Grunz, unpubl.data, 1995) and Xwnt-8 (Wilson & Hemmati Brivanlou1995; Glinka et al. 1997, 1998; Piccolo et al. 1999).

In keeping with these results, the neuralizing factorsfrom amphibian gastrula and neurula remain fully activeafter coupling to bromoacetylcellulose (Tiedemann &Born 1978) or bromocyano (BrCN–)-sepharose beads(Born et al. 1986), which prevent their uptake into thecells. In addition, a cell surface receptor for forebrain-inducing factors cannot be detected (M. John & J. Janeczek, unpubl. data, 1982).

When ectoderm is mesodermalized by treatment withactivin (see later) and then immediately combined withuntreated ectoderm, it preferentially induces hindhead,trunk and tail structures. However, when the ectodermis cultured 5–25 h before combination with untreatedectoderm, it preferentially induces forehead structures(Ariizumi & Asashima 1995). This corresponds with theresults of Okada and Hama (1943), who found a temporal change in the inducing capacity of the dorsal presumptive mesoderm (upper blastoporal lip).

Masked neuralizing factors

A masked, inactive neuralizing factor is present in theectoderm, which is induced to the neural plate(Holtfreter 1934). This factor is partially activated byfreezing and thawing and completely activated byagents that denature or dissociate protein complexes(John et al. 1984). The neuralizing factor(s) themselvesare not irreversibly inactivated by these agents. Theinduced neural plate in turn acquires neural-inducingactivity (the so-called homeogenetic inducing activity;Mangold & Spemann 1927; Waechter 1953). It is likelythat the homeogenetic induction does not depend ona transfer of inducing factor(s) from the invaginating

endomesoderm (reviewed by Tiedemann et al. 1998and references therein). To answer this question definitively, however, the spatial distribution of induc-ing factors must be known.

Because the ectoderm contains a masked neuralinducing factor, several mechanisms for neural induc-tion are possible. First, the release of the inhibition ofneuralization by factors from the mesoderm andmesendoderm could directly induce neuralization andthe activation of neural-specific genes. Second, it ispossible that by the release of the inhibition themasked neuralizing factor(s) in the ectoderm are acti-vated or a de novo synthesis of the factors is initiated.The factor(s) would then be released from the ectodermor the neural plate (homeogenetic induction), respec-tively, and activate neural specific genes in neighbor-ing cells. Finally, and most likely, both mechanismscould cooperate to reinforce the neuralization of theectoderm. This cooperation is supported by the differ-entiation and inducing capacity of the different areasof the neural plate.

The differentiation and homeogenetic inducing cap-acity of the neural plate have been investigated by heteroplastic implantation of different areas of theneural plate of Triturus cristatus early neurulae (neuralfold just elevated) into the blastocoele of early T. alpes-tris gastrulae (Einsteckmethode). The foremost trans-verse part of the neural fold differentiates frequently toforebrain and midbrain and also induces the develop-ment of forebrain and midbrain. The anterior medianpart of the neural plate develops mostly to hindbrain(rhombencephalon) and rarely to forebrain or spinalcord and induces mostly rhombencephalon develop-ment. The posterior medium part of the neural plate dif-ferentiates less frequently to rhombencephalon andmore frequently to spinal cord and induces mostlyspinal cord and muscle, which attends the spinal cord(Waechter 1953; Tiedemann-Waechter 1960). Thismuscle-inducing activity is inhibited in the embryo.

Mesodermal genes promoting neural induction

Genes that are involved in the differentiation of meso-derm, endoderm and the nervous system can act insequence, but also in parallel (reviewed by Tiedemannet al. 1996; Grunz 1999b). This unexpected redun-dancy of differentiation pathways is reminiscent ofSpemann’s principle of double insurance (‘doppelteSicherung’) in biological processes (for instance in formation of the lens; Spemann 1936). However, redun-dant gene control mechanisms are preferentiallyinvolved in the spatial and temporal fine regulation of gene expression. The interpretation of the induction results for neural, mesoderm and endoderm


differentiation is impeded by the fact that for manygenes differentially expressed in development, only thespatial and temporal distribution of the mRNA, but notof the proteins encoded by the genes, which are thereally effective molecules, are known.

The activated Xlim-1 gene has been implicated in theinduction of muscle and head neural structures in X. laevis (Taira et al. 1994) and in mice (Shawlot &Behringer 1995). A major transcriptional activator ofXlim-1 in early development is activin or an activin-likefactor (see also later). Other genes of interest in thiscontext are chordin and noggin. The chordin geneencodes a 120 kDa secreted molecule, which dorsal-izes the mesoderm (Sasai et al. 1994). The gene, whichis expressed in the dorsal mesoderm, is a secondaryresponse gene to activin. Chordin expression is activated by microinjected mRNA of the dorsal-mesodermal homeobox genes Xlim-1 and gsc (goose-coid, see later). The homeobox genes code for tran-scription factors. The homeobox is a highly conservedDNA sequence, which codes for a polypeptidesequence of 60 amino acids, the homeodomain(Gehring 1992). The homeodomain is one of thesequence motifs of transcription factors that bind toDNA.

Chordin, which is expressed in the dorsal mesoderm,inhibits ventral signals by binding directly to BMP-4,probably preventing BMP–receptor interaction (Piccoloet al. 1996). Microinjection of chordin mRNA into X. lae-vis blastomeres induces neural tissue development, asshown by histologic analysis of the isolated animal caps(Sasai et al. 1995). Chordin protein applied directly toanimal caps leads to the expression of the neuralmarker neural cell adhesion molecule (N-CAM; Piccoloet al. 1996). Xolloid, a secreted metalloprotease, inac-tivates chordin by proteolytic cleavage. When chordinin inactive chordin–BMP complexes is cleaved, activeBMP is released. Accordingly, injection of XolloidmRNA leads to ventralization at the gastrula stage. Inthe embryo, endogenous Xolloid could spatially regu-late chordin activity (Piccolo et al. 1997). Whetherchordin directly induces forebrain depends on theamount of chordin expressed between the mesendo-derm, the anterior dorsal mesoderm and the overlyingectoderm.

Noggin, like chordin, is a secreted protein, which dorsalizes the mesoderm (Smith & Harland 1992; Smithet al. 1993). Noggin also interacts directly with BMP-2/4 (Zimmermann et al. 1996). Transcription of nogginis induced by activin in animal caps (ectoderm) in thepresence of cycloheximide, an inhibitor of protein syn-thesis (Sasai et al. 1994). Microinjection of chordinmRNA into blastomeres induces the expression of theneural marker N-CAM in animal cap ectoderm; in some

cases, this occurs together with a marker for muscledevelopment. Noggin binds to extracellular heparinand diffusion of noggin may only occur when the bind-ing sites of the extracellular matrix are saturated(Zimmermann et al. 1996). The affinity of noggin forBMP is 15-fold higher than that of chordin; neverthe-less, the N-CAM-inducing activity in animal capsrequires 10-fold higher amounts of noggin comparedwith chordin (Piccolo et al. 1996).

A gene called cerberus, which codes for a secretedprotein of 270 amino acids, induces forebrain, but sup-presses formation of trunk–tail mesoderm. In X. laevis,zygotic expression of the gene starts at the blastulastage in the yolky anterior endomesoderm, includingleading edge cells of the endomesoderm invaginatingduring gastrulation. At the midgastrula stage, the geneis expressed anterior and lateral to the prechordal plate(Bouwmeester et al. 1996). This corresponds with theobservation that in Triturus a small area of the foremostpart of the neural plate, the transverse neural fold, pref-erentially develops to forebrain. In mice, the anterior visceral endoderm, which corresponds to the anteriorendomesoderm in X. laevis (the area of cerberusexpression), is important in head formation (Thomas &Beddington 1996; Rhinn et al. 1998). Cerberus can beinduced by X. laevis nodal-related factor 1 (Xnr1; seelater), but can also inhibit the expression of Xnr1, poten-tially serving as a feedback inhibitor. Cerberus proteinbinds to BMP and Wnt proteins, which promote the dif-ferentiation of ventral mesoderm (see later) and inhibitneuralization in the dorsal ectoderm. It has been pro-posed that in order for the head territory to form, thesignaling of all three factors involved in trunk develop-ment must be inhibited. A truncated form of cerberus,called cerberus short (Cer-S), interacts with and inhibitsonly the X. laevis Xnr1 gene (Piccolo et al. 1999). A cerberus-related gene is expressed in mouse embryos,but an in vivo requirement of the cerberus-like proteinfor anterior pattern formation has not thus far beenfound (Simpson et al. 1999).

Another protein, called Dickkopf (dkk-1), belongs toa new family of secreted proteins. It antagonizes Xwnt-8, which is expressed in ventrolateral gastrula meso-derm, and is involved in neuralization. Dkk1 inducesthe neural markers N-CAM and N-tubulin in animalectoderm. This induction does not, however, lead to histologic differentiation of neural tissues (Glinka et al.1998). Niehrs (2001) has proposed a two-inhibitor (anti-BMP and anti-Wnt) model for neural induction. Thismodel also explains the microcephaly observed inzebrafish in which the Wnt pathway inhibitor tcf3 ismutated (Kim et al. 2000). The follistatin protein, towhich neural inducing activity has been ascribed, hasno neural-inducing activity when it is applied in a wide


range of concentrations to animal cap ectoderm(Asashima et al. 1991a; Grunz 1996).

The proteins encoded by the genes described earlier induce only neural tissue of the forebrain. Hind-brain (rhombencephalon with metencephalon andmyelencephalon) is induced by the median anteriorand the adjacent posterior archenteron roof (invagin-ated dorsal mesoderm) of early Triturus neurulae (TerHorst 1948). When cells of the presumptive forebrainand the axial mesoderm are disaggregated and mixedin different proportions, an increase of mesodermalcells results in an increase of hindbrain and spinal corddifferentiation (Toivonen & Saxén 1968). Combinationsof partially purified forebrain and mesoderm (somites,notochord) inducing factors induce hindbrain andspinal cord development, depending on the ratio of thetwo factors (Tiedemann & Tiedemann 1964).

Basic fibroblast growth factor (see later), togetherwith other factors, has been implicated in the expres-sion of more posterior brain sections (Crox & Hemmati-Brivanlou 1995; Lamb & Harland 1995). The Wnt-3agene can alter anterior–posterior neural patterning(McGrew et al. 1995). Retinoic acid, which operates viaa nuclear receptor, has no neural-inducing activity, butretinoid signaling enhances the development of pos-terior structures (Durston et al. 1989; Ruiz i Altaba &Jessell 1991a,b; Blumberg et al. 1997). The inductionof rhombencephalon and spinal cord obviouslydepends on as yet unidentified additional factors. It ispossible that these factors act on a distinct pathwaynot involving BMP and Wnt inhibition.

Dorsoventral and anterioposterior pattern formation in the developing neural system

The interesting results on pattern formation in the devel-oping neural system can only be dealt with briefly inthis review (reviewed by Lumsden & Krumlauf 1996;Gruss & Walther 1992; Krumlauf 1994). The inducedneural plate is patterned through a series of inductiveinteractions along the dorsoventral and anterior–posterior axes. Ventral structures are induced by theunderlying mesoderm and also originate within the neuroectoderm.

The zebrafish cyclops mutation leads to a deletionof the ventral-median floor plate of the spinal cord. Themost severe deletions, however, are found in the ven-tral forebrain, especially in the midbrain, leading tocyclopia due to incomplete splitting of the eye field(Hatta et al. 1994). The cyclops locus encodes thenodal-related protein Ndr2 of zebrafish. The Ndr2 geneis first expressed in a region corresponding to theamphibian organizer (dorsal mesoderm), and isexpressed subsequently in the prechordal plate and

in the ventral neuroectoderm at the tail-bud stage. Thecyclops gene is probably autoregulated, in that cyclopsactivity appears to be required for the expression of thecyclops gene in the anterior axial mesoderm and theventral neuroectoderm. Cyclops acts as a strong neu-ralizing inducer in the X. laevis animal cap assay, prob-ably by antagonizing BMP (Rebagliati et al. 1998).Additional factors, such as sonic hedgehog (Shh), arerequired in the patterning of the anterior ventral nervoussystem. Cyclops and Shh seem to act in parallel(Chiang et al. 1996).

The mouse homeobox gene Otx2 is required for thedevelopment of the forebrain, the midbrain and theanterior rhombencephalon (up to rhombomere two).The anterior neural plate can form without expressingOtx2, but important regulatory genes, including Pax2,Wnt-1 and En (a homolog of engrailed), are notexpressed. Otx2 is the earliest homeobox gene to beexpressed in the neuroectoderm (Rhinn et al. 1998).The X. laevis homolog of Otx2 is a maternal geneexpressed at low levels. Zygotic expression starts inthe blastula stage, in a region that gives rise to pre-chordal mesendoderm, suggesting a role in the differ-entiation of the most anterior structures. At gastrulastage 10.5 (Nieuwkoop & Faber 1956), transcriptionbecomes detectable in the presumptive anterior neuro-ectoderm (Pannese et al. 1995). The Otx5 gene, iso-lated from activin-treated X. laevis ectodermal animalcaps, stimulates the formation of anterior regions andrepresses the formation of posterior structures (Kurodaet al. 2000).

The mouse En1 and En2 genes, which code for tran-scription factors and depend on Otx2 expression, arealso essential for the development of the midbrain andcerebellum. The En genes show overlapping expres-sion with the Pax2, Pax5 and Pax9 genes in the devel-oping brain. The enhancer element of the En2 gene hasbeen shown to contain multiple positive and negativeregulatory elements, including two DNA-binding sitesfor the Pax-2, Pax-5 and Pax-8 proteins. The Pax genesare most probably direct upstream regulators of the En2gene (Song et al. 1996). The FGF8 gene is expressedas a ring at the posterior border of the midbrain, justposterior to the expression of the Wnt-1 gene (Crossleyet al. 1996). The Wnt-1 protein does not induce mid-brain, but is involved in the maintenance of En expres-sion (Lumsden & Krumlauf 1996). In midbrain patternformation, the size, shape and orientation of cell popu-lations has been shown to depend on the geometry ofthe sonic hedgehog source (Agarwala et al. 2001).

The expression of the Pax6 homeobox gene in thechicken neural plate is inhibited by activin (Pituello et al. 1995) and FGF (including FGF8) from presomiticmesoderm. It has been proposed that the refinement


of dorsoventral expression of Pax is dependent on agradient of FGF signaling moving posteriorly to extendthe limit of Pax6 expression to progress caudally(Bertrand et al. 2000). Pax6 activation depends onsomitogenesis in the chicken embryo cervical cord. Themaintenance of Pax6 expression also depends onsomites (Pituello et al. 1999).

Many distinct neuronal cell types are determined bythe patterning of the nervous system. A large numberof genes, in part homologs of Drosophila melanogastergenes, are involved in the early as well as terminal dif-ferentiation of neurons (reviewed by Placzek & Furley1996; Tiedemann et al. 1998); some are reviewed here.The X. laevis X-MyT1 gene, a C2HC-type zinc fingerprotein, is involved in the primary selection of neuronalprecursor cells. The expression of the gene is positivelyregulated by the bHLH protein X-NGNR-1 and nega-tively regulated by the Notch/Delta signal transductionpathway. Inhibition of X-MyT1 function inhibits normalneurogenesis (Bellefroid et al. 1996). The Pax3 andPax7 transcription factor genes share redundant func-tions to restrict ventral neuronal identity in the spinalcord (Mansouri & Gruss 1998). They are regulated ven-trally by Shh (sonic hedgehog) and dorsally by BMP-4 and BMP-7 (Goulding et al. 1993; Liem et al. 1995,1997; Ericson et al. 1996). The expression of two setsof homeotic transcription factors is regulated in the ven-tral neural tube by a gradient of Shh activity. They definethe identity of neuronal progenitor cells. The expres-sion of five class I proteins (Pax7, Irx3, Dbx1, Dbx2 andPax6) is repressed by Shh; two class II proteins (NKx6.1and NKx2.2) are induced by Shh. It has been proposedthat cross-repressive interactions between class I andII proteins establish individual progenitor domains ofneuronal cells (Briscoe et al. 2000). The protein XNLRR-1, an X. laevis homolog of the mouse neuronal leucine-rich repeat protein, is expressed in the developing X. laevis nervous system. Its similarity to other humancell adhesion molecules implicates its involvement ininteractions at the neuronal cell surface (Hayata et al.1998). The expression of the first neuronal genes immediately proceeds to the release of the inhibitionof neuralization by neural induction (reviewed byTiedemann et al. 1998). Some precursors may have arestricted neuronal fate, but most generate neurons andastroglial cells and are at least bipotential (Cochard et al. 1995). Only a limited number of neuronal genescan be activated following dissociation and reassoci-ation of ectoderm cells (Duprat 1996).

Sox10 is the key regulator in the differentiation ofperipheral glia cells in mice, which descend frompluripotent neural crest cells. In a Sox10 mutant,Schwann cells or satellite cells are not generated.Neuronal cells in the dorsal root ganglia are formed,

but degenerate later. Sox10 controls the expression ofErbB3, which encodes a Neuregulin receptor. Neure-gulin is a member of the EGF family. Heparin sulfate atthe cell surface interferes with the free diffusion andlong range signaling of Neuregulin (Meyer & Birchmeier1995; Britsch et al. 2001).

Signal transduction in neurogenesis

Neural induction is activated via different signalingpathways, including the L-type Ca2+ channels(reviewed by Duprat 1996). An increase in internal Ca2+

mediates neural induction in P. waltl (which shows noautoneuralization). Stimulation by the L-Ca2+ channelagonist Bay K8644 induces the differentiation of neuronal and glial cell lineages. Neuronal induction ofectoderm in Holtfreter sandwiches with dorsal meso-derm (organizer) is inhibited by a Ca2+ chelator(Moreau et al. 1994).

Phorbolester (phorbolmyristateacetate (PMA, TPA)evokes large complexes of neural tissue as well somemesenchyme and melanophores in isolated T. alpestrisectoderm (which shows no autoneuralization in con-trols; Davids et al. 1987) and in X. laevis ectoderm (Otteet al. 1988), albeit at a higher concentration. Theenzyme protein kinase C (PKC), a serine–threoninekinase, translocates to the plasma membrane in PMA-treated explants, which correlates with its activation(Otte et al. 1988). The activity of PKC is enhanced inX. laevis ectoderm explants treated with neuralizing factor (Davids 1988). PKC can upregulate or down-regulate Ca2+ channels in many systems. Ca2+ chan-nels are activated by PKC either directly byphosphorylation or indirectly by downregulation of voltage-regulated Na+ and K+ channels (Alijianian et al.1991). To test the hypothesis that PMA may induceneural tissue by activating Ca2+ channels, Ca2+ levelshave been measured during treatment with PMA.Intracellular Ca2+ was transiently increased (Leclerc et al. 1995). Staurosporin, a PKC inhibitor, prevented theCa2+ increase (Moreau et al. 1994). These experimentssuggest that an activation of L-Ca2+ channels takes partin the induction of neural tissue by PMA. IntracellularCa2+ also increased when the lectin concanavalin A,which induces neuralization, was added to isolatedectoderm. The signal transducing Go protein wasexpressed simultaneously and could be colocalizedwith L-type Ca2+ channels (Pituello et al. 1991; Leclercet al. 1995). The increase in Ca2+ following the activa-tion of L-type Ca2+ channels during the differentiationof PC12 cells leads to the activation of immediate earlyresponse genes, such as c-for or Jun-B. Preliminaryexperiments have shown that a similar mechanism maybe involved in neural induction (reviewed by Duprat


1996). The molecular interactions between L-typeCa2+ channels, inhibitors of neuralization and neural-inducing factors remain to be elucidated.

N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid(HEPES), a zwitterionic buffer substance, triggers neu-ralization at a certain pH range (when the zwitterionicform prevails) in isolated ectoderm of T. alpestris(Tiedemann 1986a). HEPES activates the Na+/H+

antiport system (Altura et al. 1980). However, ethyliso-propylameloride, a specific inhibitor of the antiport sys-tem, does not inhibit neuralization in a wide range ofconcentrations (Tiedemann 1986; Hildegard Tiede-mann, unpubl. data, 1988). A change of intracellularpH is probably not sufficient to trigger neural induction.A change in the conformation of plasma membraneproteins may be involved in HEPES action.

Determination of mesoderm in the marginalzone of very early embryos without inductionby the endoderm

Following fertilization, cytoplasmic streaming (corticalrotation Ancel & Vintemberger 1948; Vincent & Gerhart1987) establishes a dorsal–ventral polarity, whereby thedorsal side of the embryo is located opposite to the random point of sperm entry. Cortical rotation leads toan upward (i.e. towards the animal pole) movement ofthe dorsovegetal subcortical cytoplasm relative to theegg surface. It has been shown by confocal micro-scopy that rotation movements are observed prior tothe appearance of a detectable parallel array of micro-tubules inside the vegetal cortex. This suggests thatthe array may be oriented by preceding cytoplasmicmovements and that the motor molecules may be dis-tributed throughout the parallel array (Larabell et al.1996). Cortical rotation modifies the animal–vegetal anddorso-anterior location of factors that are involved in thefirst steps of cell determination and thereby specifiesthe dorsoventral axis.

It has been shown that the presumptive endoderminduces mesoderm in isolated ectoderm, when pre-sumptive vegetal endoderm (later called the Nieuw-koop center) is combined with ectoderm (Ogi 1967;Nieuwkoop 1969). This experiment has been repeatedwith the same result by other investigators. Tested onectoderm, which was still competent for mesoderminduction for only 45 min, endoderm was inductive bythe 64-cell stage (Jones & Woodland 1987). Nieuwkoopconcluded from these experiments that the mesodermwithin the embryo, which differentiates from the mar-ginal zone (the region of the fertilized egg and earlyembryo between endoderm and ectoderm), is alsoinduced by the vegetal endoderm. More recent experi-ments have cast doubt on this interpretation (Gallagher

et al. 1991; Grunz 1994; Li et al. 1996). The four animalblastomeres, which were manually isolated from regu-larly cleaved X. laevis 8-cell stages as a quartet, dif-ferentiated to dorsal and dorsolateral mesodermal(notochord, somites) and (secondarily induced) neuraltissues in over 50% of cases. Ventral mesodermal tissues were not induced (Grunz 1994). The yolk-richleakage material from the dissected vegetal blasto-meres, in which the animal blastomeres came to lie for20–30 min, induced only mesenchyme and coelomicepithelium from animal cap ectoderm in control experi-ments. It is possible that dorsal mesodermal determi-nants are located in the intercellular space of 8-cellstage embryos between the animal and vegetal blasto-meres. Because no general mixing of the cytoplasmoccurs after cortical rotation, due to a stable gradientof yolk platelets, this would mean that mesodermaldeterminants are located in, and secreted from, themarginal zone adjacent to the animal blastomeres.These results suggest that the determination and earlydifferentiation of dorsal mesodermal tissues dependson factors that are located in the marginal zone aftercortical rotation. The result is in accordance with thefate map of X. laevis embryos (Nakamura & Kishiyama1971), from which it can be inferred that the dorsal animal blastomeres of the 8-cell stage provide prog-eny for part of the notochord and a smaller part of thesomites (Fig. 2). There is good agreement with the X. laevis fate map of Dale and Slack (1987), except inthe greater contribution they attribute to somites in thesecond tier of the 32-cell map. In accordance withthese experiments, B1 and C1 blastomeres (Fig. 2) ofthe 32-cell embryo can organize secondary axes, whentransplanted into host embryos (Gimlich & Gerhart1984; Gimlich 1986). This does not exclude the pres-ence of factors in the presumptive endoderm prior tomid-blastula transition, which can diffuse into the ecto-derm, are thereby diluted and induce mesoderm when

Fig. 2. Fate map of Xenopus laevis at the 32-cell stage. AfterNakamura and Toivonen (1978) from Tiedemann et al. Develop.Growth Differ. 38, 234 (1996).


the ectoderm is combined with the presumptive endo-derm (see later regarding endoderm and mesoderminduction at different concentrations of activin (vege-talizing factor)).

To analyze the effect of the vegetal presumptiveendoderm or of animal ectoderm on mesoderm differ-entiation, the marginal zone alone, the marginal zonetogether with the vegetal region or the marginal zonetogether with the animal region were isolated from dif-ferent stages of A. mexicanum embryos. The stageswere determined by measurement of the diameter ofanimal pole cells (Tiedemann 1993). Ambystoma mexi-canum is well suited for these experiments, becauseof its large eggs and slow development. No differenti-ation occurred in the isolated marginal zone of veryearly blastula-stage embryos. Unexpectedly, when themarginal zone was isolated together with the animalectodermal region from late morulae (the earliest stagein which the operation could be done), dorsal (noto-chord), dorsolateral (somites) and ventral (renal tubulesand mesothel) mesoderm differentiated. Endoderm didnot develop. When the marginal zone was isolatedtogether with the vegetal presumptive endodermalregion, notochord differentiated in the late early blas-tula stage and somites developed not before the latemid-blastula stage; that is, the animal region must bein contact with the marginal zone up to this stage.Ventral mesoderm did not develop. Neural tubes wereinduced by the primarily induced mesoderm in somesamples of the animal region–marginal zone explants.Autoneuralization was not observed in the isolated animal region. Blood precursor cells developed in thelate blastula stage in the animal region–marginal zoneexplants (Tiedemann 1993). It has been shown that, inaddition to BMP (see later), factors are expressed inthe ectoderm of X. laevis gastrulae that stimulate erythropoiesis in the ventral mesoderm (Maéno et al.1994).

It has been suggested from the above-describedresults that in the determination of mesoderm in A.mexicanum, factors from the ectoderm could beinvolved. It is possible, however, that a factor (probablyactivin) could be present in a too-high relative con-centration in the marginal zone when the ectodermalregion is dissected from the marginal zone–vegetal(endodermal) region explants. This would explain why,in the marginal zone–vegetal region explants, noto-chord, but no ventral (renal tubules and mesothel) and(until the late mid-blastula stage) lateral (muscle)mesodermal tissues are induced. In contrast, ventro-lateral tissues are induced in marginal zone–animalregion explants and in animal–vegetal region combi-nates. Ventrolateral tissues are induced at a relativelylow concentration of activin, together with FGF and

BMP (Tiedemann 1993; Hildegard Tiedemann, unpubl.data, 1995).

Mesoderm and endoderm induction and differentiation: Genes and factors

Maternal secreted factors and maternal transcriptionfactors have been found in all three germ layers. In theearly gastrula, after the so-called mid-blastula transi-tion of X. laevis (Newport & Kirschner 1982), a morecomplex pattern of gene expression emerges. After themid-blastula stage, the transcription of many mRNA isenhanced. A small amount of high molecular mRNA-like RNA, however, is already synthesized duringcleavage (Nakakura et al. 1987). Most eukaryoticgenes contain several enhancer (and silencer)sequences for transcription factors in their regulatoryregions. As well as the DNA-binding domains, factorshave protein-binding domains, indicating allostericinteractions (Monod et al. 1963; Wyman 1964) withneighboring proteins.

Activins, vegetalizing factor, activin-like factorsand fibroblast growth factors

A protein isolated from chicken embryos has beenfound to induce mesoderm and endoderm develop-ment and is called vegetalizing factor. The factor hasbeen extracted with phenol, which dissolves protein,whereas RNA from the aqueous phase has no induc-ing activity (Tiedemann & Tiedemann 1956a,b). Theprotein has a molecular weight of 26–30 kDa and formsa homodimer with subunits of 13 kDa (Geithe et al.1981; Schwarz et al. 1981). It has been purified �106-fold. The final purification was achieved by serialreversed phase (RP)-HPLC on different stationaryphases (Plessow et al. 1990). Reduction with formicacid preferentially splits an exposed disulfide bond(Daopin et al. 1992; Schlumegger & Grütter 1992)between the two subunits and is in part reversible.Reduction with mercaptoethanol splits all disulfidebonds and leads to an irreversible loss of inducingactivity. The factor induces all dorsal, dorsolateral andventral mesodermal tissues in T. alpestris and X. lae-vis embryos in a dose-dependent manner (Grunz 1983;Plessow et al. 1990), including primordial germ cells(Kocher-Becker & Tiedemann 1971) and blood cells.In the presence of high concentrations of the vegetal-izing factor, endodermal tissues (intestine, liver, pan-creas) have been identified after 3–7 weeks of cultureto obtain good histologic differentiation in sandwichexplants of T. alpestris (Kocher-Becker & Tiedemann1971) and in X. laevis animal cap explants (Asashimaet al. 1991a). Mesodermal patterning depends on


secondary inducing interactions by additional factors(Asahi et al. 1979; Minuth & Grunz 1980). When thevegetalizing factor is implanted at high concentrationsinto the blastocoele of an early Triturus alpestrisgastrula, the already invaginated endoderm and mesoderm spreads over the induced ectoderm (exovagination, which should not be confused with exo-gastrulation), due to a change of cell affinities (Kocher-Becker et al. 1965). This change is mediated by theearly activin response gene gsc (Niehrs et al. 1993).The change of cell affinities has been measureddirectly in dissociated and activin-induced cells by theirpatterns of sorting out following reaggregation (Kurodaet al. 1999).

The vegetalizing factor can be purified by chromato-graphy on heparin-sepharose (Born et al. 1987).Heparin-binding growth factors (bFGF and acidicfibroblast growth factor; aFGF) have therefore beentested for their effects on induction. It has been shownthat both FGF can induce trunk and tail structures withthe ventral mesodermal tissues, including blood islandsand heart muscle surrounded by an endothel-linedpericardial cavity, as well as somites (Knöchel et al.1987; Slack et al. 1987; Grunz et al. 1988; Knöchel et al. 1989a). eFGF with a secretory signal sequenceis a more potent inducer of mesoderm than bFGF(Isaacs et al. 1994). Whether FGF directly induceneural tissue in amphibian embryos is a controversialquestion (Doniach 1995). In some T. alpestris animalcap experiments, large masses of neural tissue with-out mesoderm have been induced by a very high con-centration of recombinant bFGF (Tiedemann et al.1994). However, this is not a physiologic process. Inchicken embryos, the gene ERNI, an early responsegene from Hensen’s node (equivalent to the amphib-ian organizer), has been used to show that FGF8 may‘sensitize’ the epiblast to the expression of later neuralmarkers before gastrulation. However, FGF signals arenot sufficient to generate a whole nervous system (Streitet al. 2000). Activation of the extracellular signal-regulated protein kinase (ERK) in X. laevis blastulae isgenerated by endogeneous FGF signaling. Immuno-staining suggests that FGF protein can diffuse over several cell diameters (Christen & Slack 1999).

The FGF are monomers and have a much higheraffinity for heparin than the vegetalizing factor, whilemembers of the transforming growth factor (TGF-�)family are closely related in their structure to the vegetalizing factor. Dawid and colleagues and Tiede-mann and colleagues (Knöchel et al. 1987; Rosa et al.1988; Knöchel et al. 1989b) have shown independentlythat TGF-�1 (2 µg/mL) induces endothel (in someexplants arranged in a capillary-like network), bloodprecursor cells, mesenchyme and strands of seg-

mented blastema tissue. TGF-�2 (1–3 µg/mL) inducesall dorsal and ventral mesodermal tissues. Hemato-poietic progenitor cells can be identified by the expres-sion of embryonic �-globin (Oschwald et al. 1993).

In 1989, Asashima and coworkers discovered thatactivin A, a member of the TGF-� superfamily, has amuch higher mesoderm-inducing capacity (80% induc-tion at 1 ng/mL; Asashima et al. 1989; Asashima et al.1990a). Activin A was originally isolated from folliclefluid as a gonadal hormone that stimulates follicle-stimulating hormone secretion. It is identical to the erythroid differentiation factor (Murata et al. 1988) andinduces all mesodermal tissues (Ariizumi et al. 1991)and endoderm (Jones et al. 1993) in a dose-dependentmanner. Together, Asashima and colleagues andTiedemann and colleagues have demonstrated that thevegetalizing factor is closely related to activin(Asashima et al. 1990b). Both factors are inhibited byfollistatin (Asashima et al. 1991a). Partial sequencingidentified the vegetalizing factor as an activin Ahomolog (Tiedemann et al. 1992). A factor isolated fromX. laevis transformed fibroblasts (XTC factor; Smith et al. 1990) and a factor isolated from the amniotic fluid(Chertov et al. 1990) have also proved to be eitheractivin or activin homologs.

The FGF and activins or a related factor areexpressed as maternal proteins (Kimelman et al. 1988;Asashima et al. 1991b; Isaacs et al. 1992). Activin A(�A/�A), activin B (�B/�B) and activin AB (�A/�B) and fol-listatin in abundance have been isolated from X. lae-vis eggs and blastulae. The activin proteins are in partpresent as a complex with follistatin, which inhibits theactivins. Activin A and activin B have equivalent induc-ing activity (Asashima et al. 1991b; Fukui et al. 1994).Activin mRNA is not present in these early stages.Zygotic activin B is transcribed in X. laevis after the mid-blastula transition stage, whereas zygotic activin A isnot transcribed before the end of gastrulation (Thomsenet al. 1990; Dohrmann et al. 1993). Activin A is pre-dominantly transcribed in the follicle cells surroundingX. laevis oocytes (Dohrmann et al. 1993; Rebagliati &Dawid 1993). In mice, activin A and activin B mRNAare present in the granulosa cells of the follicles, butthey are also present at lower levels in eggs and pre-implantation embryos (Albano et al. 1993).

The temporal distribution of activin mRNA in folliclecells, oocytes and embryos of Oryzias latipes (teleostfish, Japanese medaka) is very similar to the distri-bution of the activin mRNA in X. laevis. The spatial and temporal distribution of activin protein has beenmeasured by a monoclonal antibody. The cytoplasmand germinal vesicles of large oocytes as well as thefollicle cells were positively labeled (Ge et al. 1993;Wittbrodt & Rosa 1994). Early medaka embryos were


uniformly stained from the 1-cell stage until at least theblastula stage. Two activin dominant-negative mutantswere generated. Injection of RNA from one of thesemutants into a medaka blastomere led to a dose-dependent loss of axial mesoderm. This mutant actsat the protein level and blocks the interaction of mater-nal activin protein with its receptor. The second mutantinteracts at the mRNA level. It was found to deplete theactivin pool when cotranslated with wild-type activin,although axis formation was not disturbed. This sug-gests that zygotic activin mRNA is not necessary formesoderm differentiation (Wittbrodt & Rosa 1994).Activin also induces axial structures in the hypoblastof chicken embryos (Mitrani et al. 1990).

The X. laevis Vg1 gene, which is related to the activingenes, encodes a maternal mRNA that is localized inthe vegetal region of oocytes and embryos (Tannahill& Melton 1989). Vg1 belongs, like activins, to the TGF-� superfamily and displays homology in the C-terminal region, on which the inducing activitydepends (reviewed by Tiedemann et al. 1995). The pro-tein is abundant in embryos as a precursor, which hasno inducing activity. The processed active form can-not be detected. Tannahill and Melton (1989) propose,as a hypothesis, that localized processing of the pre-cursor is the limiting step. The gene constructs Bm-Vg1or Act-Vg1, which code for processed proteins, showsimilar activities to the activins (Thomsen & Melton1993). In zebrafish, an ortholog of X. laevis Vg1 is alsopresent as an unprocessed precursor. The zebrafishprecursor is expressed to some degree as a matureprotein when overexpressed in X. laevis embryos(Dohrmann et al. 1996). A X. laevis Vg1 RNA-bindingprotein is expressed in the vegetal region of X. laevisoocytes and early embryos. A homologous RNA-binding protein (KOC) in mammalian embryogenesisis highly expressed in the gut, pancreas and kidneyand overexpressed in pancreatic cancer (Mueller-Pillasch et al. 1999). It would be interesting to knowwhether Vg1 is processed at later stages of develop-ment.

Follistatin, proteoglycans, inducing factors andreceptors

Fukui et al. (1999) have measured the uptake and local-ization of labeled activin and follistatin synthesized infollicle cells, as well as vitellogenin synthesized in thematernal liver, in vitellogenic oocytes and their mutualinteractions by surface plasmon resonance analysis in vitro. The data are consistent with an independenttransport of the three proteins, formation of anactivin–follistatin complex and binding of the complexto vitellogenin. The catabolism of yolk platelets through-

out embryogenesis (also in blastomeres) could releasethe activin–follistatin complex (Karasaki 1963). Activinmay be more tightly bound to its cell surface receptorsthan to follistatin (see later). Whether activin is releasedfrom follistatin by proteolytic cleavage (as is BMP fromthe chordin complex, see earlier) is not known (Fukuiet al. 1999).

The secreted activins are bound to cell surfacereceptors. Activin receptors are heteromeric complexescontaining at least one molecule from each of two sub-families (I and II) of receptor serine–threonine kinases(Attisano et al. 1992; Heldin et al. 1997; Massagué1998). A cloned type II receptor (ActR-lI�) has beenshown to have the highest affinity for activin (dissoci-ation constant KD ~0.1 nM). The activin–follistatin com-plex has a lower binding affinity (KD �2.4 nM; Fukui et al. 1999).

An activin receptor of X. laevis has been shown to be a maternal protein (Chang et al. 1997). Animalblastomeres from the 8-cell stage of X. laevis (isolatedby treatment with Ca2+- and Mg2+-free solution) can beinduced to form mesoderm by treatment with activinfor 30–60 min, followed by treatment with a large excessof follistatin to inactivate any residual activin, which isnot removed by washing the cells after treatment withactivin (Kinoshita et al. 1993). This shows that functionalactivin receptors are present in X. laevis at the 8-cellstage. Receptors for FGF are expressed very early indevelopment in the animal region and marginal zone(Ding et al. 1992), as is the case for activin receptors.

In embryos, the availability of activins, TFG-�, FGFand follistatin also depends on proteoglycans of theextracellular matrix and the cytoplasm, which can formlow-affinity complexes with the factors. The vegetaliz-ing factor can bind to an acidic proteoglycan in thesupernatant of embryo homogenates, inhibiting the factor (Born et al. 1969, 1972b). Polyvinylsulfate com-petitively displaces the acidic proteoglycan, therebyactivating the factor (Tiedemann et al. 1969). Proteolyticdegradation of the protein core inactivates the proteo-glycan (Born et al. 1972a). Cell surface proteoglycanscan, in contrast, reduce their diffusion from three to twodimensions through binding the factors (Richter &Eigen 1974) and thereby increase the local concen-tration of the factors in the vicinity of their cell surfacereceptors. Betaglykan, a broadly distributed membraneproteoglycan with heparin- and chondroitin-sulfatechains attached to a 100 kDa core protein, binds TGF-� and presents the factor to the kinase subunit of theTGF-� receptor (López-Casillas et al. 1993). In rat follicular granulosa cells, where differentiation isenhanced by activin, heparan sulfate chains have beenshown to associate with follistatin (Nakamura et al.1991). The action of activins is also modulated by


follistatin in these cells. Other experiments have shownthat proteoglycans are needed for mesoderm forma-tion. Removal of heparin sulfate from proteoglycans inectoderm explants by heparinase inhibits induction ofXbra (brachyury) by activin and Xbra and cardiac actinby FGF (Itoh & Sokol 1994).

Because of the interaction of activin and follistatinwith one another and with proteoglycans, the concen-tration of active activin is not identical to the totalamount of activin in the cells. Activation of gene expres-sion by activin depends on proteins in the signalingpathway (see later) from the plasma membrane activinreceptors to nuclear factors, which bind to a DNAsequence in the enhancer region of the target gene,called the activin response element (ARE). A gene con-struct of the enhancer including one or multiple ARE,the promoter and a reporter gene (for example theluciferase gene) can be used as a probe for activeactivin. Such a probe derived from the activin-depen-dent gsc gene promoter has been microinjected intoeach single blastomere of 32-cell stage X. laevisembryos. Luciferase activity was measured duringearly gastrulation and therefore represents the activitydistribution in early gastrulae. A uniformly high activitywas found in all cells of the third and fourth tier (Fig. 2), which represents the vegetal and most of themarginal region. Low activity was found in the dorsalmost blastomeres of the second tier and very little activity was found in the other animal blastomeres(Watabe et al. 1995). A graded distribution in earlierstages is not excluded. The presence of ARE in activin-dependent genes supports the regulatory function ofactivin or activin-like factors in early stages of embryodevelopment.

Three arguments against activin being a mesoderm-and endoderm-inducing factor have been proposed.Truncated dominant-negative receptors disturb meso-derm induction by activins. However, activin receptorsare not specific; they form signaling complexes withother factors of the TGF-� family. More recently, areceptor has been designed that selectively blocks thefunction of activins, but not Vg1 (Dyson & Gurdon1997). The receptor consists only of the ligand bind-ing extracellular domain and is therefore likely to besecreted. Injection of truncated receptor mRNA in X. laevis embryos at the 4-cell stage represses theactivin-dependent pan-mesodermal gene Xbra (Xeno-pus brachyury) at the late blastula stage to 20% of thewild-type level. The inhibition then declines andreaches the wild-type level between the gastrulastages 10.5 and 11 (Nieuwkoop & Faber 1956). Thetruncated receptor obviously inhibits mesoderm induc-tion. The decline of the inhibition could depend on proteolytic degradation of the truncated receptor,

which is not inserted in the plasma membrane. In addi-tion, the affinity of the secreted receptor for activin couldbe lowered. However, it is most likely that other factors,especially the nodal-related factors, maintain andenhance mesoderm differentiation in gastrulae andlater stages (see later).

Injection of human follistatin mRNA up to 2 ng intofertilized X. laevis oocytes does not block dorsal or ven-tral mesoderm formation. Larger amounts of follistatinRNA were toxic (Schulte-Merker et al. 1994). Because follistatin protein binds and inactivates activin protein,but does not bind to processed Vg1 derived from a Vg1construct, Schulte-Merker et al. concluded that activindoes not induce mesoderm. Because follistatin bindsto BMP-4 (Fainsod et al. 1997) and BMP-7 (osteogenicprotein 1; Yamashita et al. 1995), and probably also toBMP-2, it could be concluded with the same justifica-tion that BMP-4 is not involved in ventral mesoderminduction. However, this is very unlikely. Xenopus lae-vis follistatin RNA, which is not toxic and therefore canbe injected in higher amounts (up to 8 ng), inhibitsactivin-dependent genes. Ventral expression of thepan-mesodermal marker Xbra is strongly inhibited andthe dorsal expression is inhibited in part. Xwnt-8, whichis expressed in the ventral and lateral marginal zone,is more strongly repressed than gsc in the dorsal orga-nizer mesoderm. In addition to activin, BMP are alsoinhibited (Marchant et al. 1998). Furthermore, it isunlikely that from 2 ng follistatin mRNA, which wasinjected in fertilized eggs, enough follistatin is trans-lated up to the 8-cell stage, when activin can bind toits receptor (see earlier). It is not known in which stageenough follistatin protein is synthesized to compete withthe maternal activin protein. This could be the reasonthat a relatively large amount of follistatin mRNA isneeded to see an effect.

Deletion of the activin A and/or activin B genes inmice by gene targeting does not lead to the expectedmalformation of mesodermal tissues and early deathof embryos (Matzuk et al. 1995). It is likely that theembryos could be rescued by activin from the ovarianfollicles of their mothers (see earlier). This cannot betested, however, because homozygous activin-deficientmice die after birth.

Dorsalization and ventralization of mesoderm

Activins generate only a coarse pattern in presumptivemesoderm and endoderm. Additional factors, whichdepend in part on activin, dorsalize or ventralize themesoderm. Yamada (1940) was the first to discover adorsalizing activity in the marginal zone. The dorsaliz-ing factors chordin and noggin have already been dis-cussed. Xwnt-8 is a member of the large family of


Wnt/wg secreted glycoproteins. Xwnt-8 is most abun-dant in the future ventrolateral mesoderm of X. laevisgastrulae and is probably involved in the specificationof ventral and somitic mesoderm (Christian et al. 1991;Hoppler et al. 1996). Xwnt-8 by itself cannot inducemesodermal tissues in isolated ectoderm. However, itcan be induced in ventral mid-blastula ectoderm byactivin (Christian & Moon 1993). Xwnt-8 is also inducedby FGF in animal caps (Christian et al. 1991). An intactFGF-signaling pathway (but not FGF signaling) isneeded for activin signaling (Cornell & Kimelman 1994).

Overexpression of Wnt-8 prior to the mid-blastulastage by injection of Wnt-8 mRNA into ventral blasto-meres at the 16-cell stage induces a secondary axiswith a second gsc domain (McMahon & Moon 1989).This result led to the supposition that Wnts are alsoinvolved in the dorsal �-catenin signaling pathway (seelater). The embryonic Wnt of the dorsal pathway is notyet known, therefore this pathway is commonly calledthe Wnt-like �-catenin pathway.

Activation of genes in the marginal zone (the presumptive mesoderm)

The pan-mesodermal Xbra (brachyury) T-box gene canbe activated in animal ectoderm by activin (Smith et al. 1991). At the blastula stage (zygotic) eFGF andXbra can activate expression of each other, suggest-ing that they form a regulatory loop. In the gastrula,maintenance of Xbra expression requires FGF signal-ing (Isaacs et al. 1994; Schulte-Merker & Smith 1995).In high concentration, gsc appears to directly repressthe transcription of Xbra (Latinkic & Smith 1999). Thegenes gsc, Mix.1 (Rosa 1989) and Mix.2 (Vize 1996),which are expressed in the mesoderm and part ofendoderm, belong to the immediate early activinresponse genes. Their expression is independent ofprotein synthesis. Goosecoid shows a strong super-induction by activin and cycloheximide (Tadano et al.1993). Xlim 1 is also activated by retinoic acid (Taira etal. 1994). X posterior (Xpo) and Xhox 3 are other genesexpressed in posterior mesoderm; they are preferen-tially activated by FGF (reviewed by Asashima 1994).Activin and activin-related factors also stimulate theexpression of the immediate response genes XFD-1/1�and XFD-4/4� (Dirksen & Jamrich 1992; Knöchel et al.1992; Ruiz i Altaba & Jessell 1992; Köster et al. 2000),which belong to the fork head multigene family of tran-scription factors. The DNA-binding domain of the forkhead proteins has the shape of a winged helix(reviewed by Kaufmann & Knöchel 1996). The XFD 1/1�gene is expressed in the presumptive dorsal mesodermof the blastoporal lip. Transcripts can be detected laterin the notochord and in the neural floor plate, probably

as a direct response to signals emitted from the noto-chord or to a common origin of notochord and floorplate cells. Like gsc (Fainsod et al. 1994), the XFD 1/1�gene is downregulated by BMP (Clement et al. 1995;see also later). The XFD-4/4� gene is expressed in thedorsolateral mesoderm, but not within the dorsalblastoporal lip. This is probably due to an activin-induced activation of inhibitory factors. XFD-1 over-expression inhibits an XFD-4 promoter–reporter gene(Köster et al. 2000). In contrast to other XFD genes,XFD-2/2� genes are initially transcribed within the animal hemisphere in the mid-blastula stage and sub-sequently in the marginal zone (Lef et al. 1994). XSIP1,a two-handed zinc finger protein, shows marked simi-larities to the mouse Smad (see later) interaction pro-tein 1 (SIP1) and may be a transcriptional repressor ofan activin-dependent signaling pathway (Eisaki et al.2000).

The spatial expression of Xegr-1, a gene encodinga zinc finger protein, is similar to that of the Xbra gene.In contrast to Xbra transcription, however, transcriptionfactors to be phosphorylated and activated by MAPK(like the Ets proteins, which interact with serumresponse factor (SRF) to form ternary complexes) arerequired and sufficient for Xegr-1 gene expression(Panitz et al. 1998 and references therein).

Activin response elements, activin response factors, Smad and Fast proteins

The ARE of the XFD-1/1� gene (Howel & Hill 1997), thegsc gene (Watabe et al. 1995) and the Mix.2 gene(Huang et al. 1995) are located within introns or at the5� upstream regions. The XFD-1 gene contains an addi-tional activin-activated element (AAE) in the upstreamregion, which is indirectly addressed by activin proba-bly requiring de novo synthesis of a yet unknown addi-tional factor (Kaufmann et al. 1996; Friedle et al. 1998).Separate sequence elements for the Wnt-like �-cateninsignaling pathway (see later) have been found in thegsc enhancer proximal to the ARE (Watabe et al. 1995).Wnt, however, does not directly interact with the gscenhancer; instead, the homeobox gene twin (twn) medi-ates the induction of gsc (Laurent et al. 1997). Thehomeobox gene siamois, which is a major mediator ofthe Wnt/�-catenin signaling pathway and is closelyrelated to the twin gene, can also activate gsc (Carnacet al. 1996). In embryos hyperventralized by UV irradi-ation, cortical rotation does not take place (Scharf &Gerhart 1980). In these embryos, a Xtwin reporter geneis hyperinduced in vegetal pole cells. This suggests thatcortical rotation distributes determinants of the Wnt/�-catenin pathway to the dorsal side, including the dor-sal marginal zone of the embryo (Laurent et al. 1997).


The ARE of the homeobox Xlim-1 gene is located inthe first intron of the gene (Rebbert & Dawid 1997).Xlim-1 is also activated by the proteins encoded by theAct-Vg1 construct and by nodal (see later), but not byBMP-4, Wnt-8 or members of the Wnt/�-catenin sig-naling pathway (see later). The ARE in the 5� flankingregions of the gsc and Mix.2 genes function as ‘clas-sical’ enhancers and transcription of these genes isstimulated by activin. However, Xlim-1 contains a con-stitutive promoter in the 5� flanking region, which isunresponsive to activin. The ARE in intron I acts as asilencer of the gene. Binding of activin to the ARE ofXlim-1 mediates a stimulation of transcription, which ishigher than the constitutive transcription of the gene inthe absence of the ARE (Rebbert & Dawid 1997).

The lim-5 genes of X. laevis and zebrafish are closelyrelated to the Xlim-1 gene in sequence, but not inexpression pattern. The Xlim-5 gene is activated cellautonomously in the entire ectoderm of early gastrulaeand its expression is suppressed by activin. Duringneurulation, the genes are rapidly restricted to the anterior region of the developing neural plate (Toyamaet al. 1995).

The ARE of the different activin-responsive genesshow low sequence homology. A number of proteinsinteract, however, in the formation of multiproteinactivin response factor complexes and contribute to thespecificity of activin signaling. After the activation of thecell surface receptors by binding of TGF-� factors,Smad proteins are translocated to the nucleus, wherethey participate in DNA binding and gene activation orrepression. Based on sequence homology and bio-logical activity, three groups of Smads can be distin-guished: (i) receptor specific Smads; (ii) commonmediator Smads; and (iii) inhibitory Smads (Massagué1998; Whitman 1998). All members contain two highlyconserved domains in the N- and C-terminus, sepa-rated by a proline-rich region of variable sequence andlength. Binding of activins to type II receptors and, inturn, activation of type I receptors results in the phos-phorylation of Smad2. The C-terminal region of Smad1can probably act as an activator or inhibitor, depend-ing on the region of expression in the embryo (Muelleret al. 1999). In later stages of zebrafish development,Smads determine the regionally restricted effects ofTGF-� signaling.

Additional factors take part in the formation of tran-scription complexes. A multiprotein activin responsefactor (ARF) binds to the ARE of the endodermal-mesodermal Mix.2 gene. This ARF has been shown tobe composed of the novel transcription factor Fast-1and Smad 2 or Smad3 and Smad4. Fast-1 is a nuclearprotein containing an N-terminal fork head bindingdomain and a C–terminal Smad interaction domain,

which is responsible for activin-regulated associationwith the Smads. Fast-1 recognition of the ARE is essen-tial for ARF binding. Smad binding enhances the bind-ing and regulatory activity of the ARF (Chen et al. 1996;Yeo et al. 1999).

Ventralizing genes in early X. laevis embryos, thebone morphogenetic factors and the Xvent genes

Knöchel and colleagues have shown that the BMP-2and BMP-4 genes are transcribed in X. laevis oocytesand embryos (Köster et al. 1991; Plessow et al. 1991).The BMP belong to the TGF-� superfamily and arerelated to activin. The BMP proteins are, besides theirrole in the inhibition of neuralization, the main ventral-izing factors. The BMP-2 gene is upregulated duringoogenesis. Maternal transcripts are abundant in cleav-age stages, but decline rapidly during gastrulation.Zygotic transcription of BMP-2 starts in early neurulae.The transcripts are localized to neural crest cells, olfac-tory placodes, pineal gland and the heart anlage.Maternal BMP-2 transcripts show some enrichmentwithin the animal region. Zygotic BMP-4 transcriptsincrease in abundance after the late blastula stage(Köster et al. 1991; Yamagishi et al. 1995) and can over-ride dorsalizing signals (Dale et al. 1992; Jones et al.1992; Fainsod et al. 1994; Graff et al. 1994; Clement et al. 1995). A BMP-4 protein gradient declining fromventral to dorsal may contribute to the proper forma-tion of distinct mesodermal tissues (Dosch et al. 1997).

The Xvent-1 and Xvent-2 subfamilies of the Xventgenes are involved in ventral BMP signaling and canmediate the regulatory effects of BMP-2/4. Xvent-1 andXvent-1B are expressed in the ventral marginal zoneof early X. laevis gastrulae (Gawantka et al. 1995, 1998;Rastegar et al. 1999). Xvent-2 and Xvent-2B areexpressed in the ventral marginal zone, as well as inthe lateral parts of the mesoderm, with the exceptionof the most dorsal cells (Onichtchouk et al. 1996;Rastegar et al. 1999). Xvent-2B is directly activated byBMP-2/4 in the absence of de novo protein synthesis.Xvent-1B does not directly respond to BMP-2/4, but isactivated by Xvent-2B. Cycloheximide treatment to prevent protein synthesis has shown that Xvent-2B byitself is not sufficient to activate transcription of theXvent-1B gene; additional factors synthesized after themid-blastula transition are required (Rastegar et al.1999).

XFD-1� contains an AAE that is both necessary andsufficient for transcriptional activation of reporter genesin animal cap explants. This AAE is also active withinvegetal explants in the absence of added inducers(activin is present in the vegetal region, Watabe et al.1995; see earlier). An additional inhibitory response


element, acting as a silencer, prevents the transcrip-tion of the XFD-1� gene in the ventral-vegetal region ofthe embryo in vivo. This element is located upstreamof the AAE, responds to BMP-2- and BMP-4-triggeredsignals and overrides the activation by activin.Accordingly, the temporal activation and spatial restric-tion of XFD-1� to the dorsal mesoderm is regulated bythe antagonistic action of activin or activin-related factors and BMP (Kaufmann et al. 1996). It has beenfurther shown that Xvent-1 mediates the suppressionof XFD-1� by using its BMP inhibitory element. Therepressor domain of the Xvent-1 protein is localized inthe N-terminal region (Friedle et al. 1998).

The transcription complex of the Xvent-2B gene,which is induced by BMP-4, contains a distal bindingregion for Smad1 with two GCAT motifs and proximalAGnC binding sites for Smad4. The latter is conservedin other TGF-� response elements. Ectopic expressionof Smad1 and Smad5 mimics BMP-4 induction of ventral mesoderm. Smad2 mimics induction of dorsalmesoderm by activin and nodal-related factors (seelater). Phosphorylation of the receptor-specific Smadscauses hetero-oligomerization with Smad4 and trans-location to the nucleus (Henningfeld et al. 2000 and ref-erences therein). Other transcription factors conferadditional DNA-binding specificity for activation byBMP-4. The zinc finger protein OAZ binds directly toSmad1 to activate the Xvent-2 enhancer (Hata et al.2000). The dorsal gsc gene can downregulate BMP-4in a regulatory loop after the mid-blastula stage andvice versa, depending on the concentration of the geneproducts (Fainsod et al. 1994).

BMP-2/4 shift the response to activin from dorsal toventral mesoderm (Jones et al. 1992; Clement et al.1995), whereas high concentrations of activin inhibit theformation of ventral mesoderm. Activin at low concen-tration can, in contrast, enhance Xvent-2 (Xom) tran-scription (Ladher et al. 1996), which mediates theventralization by BMP (see earlier). This implies that thetype of tissue induced depends on the ratio of activinand BMP and the timing of expression of the genes. Itcan in part be explained in this way, that the type ofmesodermal tissues induced in animal caps dependson the concentration of activin, because the animal capcontains BMP protein.

The organization of the BMP-4 gene enhancers hasalso been studied. Enhancer–promoter studies of theBMP-4 regulatory regions with luciferase reportergenes have revealed enhancers within the upstreamregion and within the second intron, which interact witha proximally located indispensable promoter. TheBMP-4 gene, as other genes, is regulated by anautoregulatory loop. The autoactivatory enhancer ele-ments are activated by BMP-4 and BMP-2. BMP-2

could therefore induce zygotic BMP-4 in embryos (Metzet al. 1998).

Endoderm: Genes and differentiation

The initial direct determination of mesoderm in the mar-ginal zone does not negate the exchange of proteinsbetween endoderm and mesoderm in later stages. Thehomeobox gene milk (mix-like) is closely related toMix.1 and Mix.2 (see earlier; Rosa 1989; Vize 1996).Like Mix.1/2, milk can be activated by activin in animalcaps and is expressed in the marginal zone (pre-sumptive mesoderm) and the vegetal endoderm ofearly gastrulae. During gastrulation, its expression isrestricted to the endoderm. Overexpression of milk inthe marginal zone represses the expression of meso-dermal genes and increases the expression of endo-dermin, an endodermal gene (see later). In the embryo,milk could prevent the extension of mesoderm into theendoderm (Ecochard et al. 1998). The mesoderm dor-salizers chordin and noggin (Smith & Harland 1992;Sasai et al. 1994) can also induce part of the endoderm.Chordin is expressed not only in dorsal mesoderm, butalso in endoderm bottle cells adjacent to dorsal meso-derm. It induces endodermin in the embryo and in animal caps, starting at the dorsal blastoporal lip, andis expressed almost exclusively at the tail-bud stagein the endoderm (Sasai et al. 1996). That the vegetal-izing factor (activin homolog) induces endoderm athigh concentrations has been mentioned previously(Kocher-Becker & Tiedemann 1971).

Mixer (Mix-like endodermal regulator) is anotherhomeobox-containing transcription factor involved inendoderm development. The gene is expressed exclu-sively in endoderm and can be turned on in animalectoderm by activin-related factors. Mixer is requiredfor the expression of the activin-inducible but not theFGF-inducible endodermal genes Xsox 17� and Xsox17� (Hudson et al. 1997). In the embryo, all three genescan be expressed independently of one another andmixer can maintain the expression of the sox 17 genes(Henry & Melton 1998). GATA5, a zinc finger tran-scription factor, is expressed in the vegetal region ofX. laevis embryos from the early gastrula stage on.GATA5 and GATA4 are potent inducers of endodermalmarker genes in animal caps. GATA5 can be inducedin the animal cap by high concentrations of activin. Inthe embryo, the GATA5 gene is likely to be preferen-tially induced by nodal-related factors (see later;Weber et al. 2000).

Explants of presumptive endoderm isolated frommid- to late blastulae and cultured until the tail-budstage express endodermal markers differentially.Expression of the more anterior pancreas marker


XlHbox-8 relies on the B-Vg1 construct, activin andFGF, while expression of the more posterior intestinalfatty acid binding protein (IFABP) does not. This sug-gests that a prepattern exists in the developing endo-derm. Cortical rotation is required for the expression ofmore anterior markers, but not for those that are moreposterior (Henry et al. 1996). The developmental biology of the pancreas has been reviewed by Slack(1995). Key events of pancreas formation can beinduced in gut endoderm by ectopic expression of pan-creatic regulatory genes. This group includes the tran-scription factors Pdx-1 and ngn13 and the genesHlxb9, Pax4, NKx2.2 and NKx6.1, which are expressedin the insulin-producing �-cells (Grapin-Botton et al.2001). Insulin is already expressed in mouse pancreasby the 20 somite stage (Gittes & Rutter 1992). The dif-ferentiation of endodermal tissues takes place at thelater stages and depends on adjacent mesoderm(Mangold 1949; Okada 1954).

The Wnt/�-catenin signaling pathway

Wnt proteins are widely distributed and play a role inmany signaling pathways in addition to embryogene-sis. Inappropriate activation of Wnt signaling has beenimplicated in human cancers (reviewed by Peifer &Polakis 2000). The so-called Wnt-1/wg class of Wnt pro-teins can induce the formation of a secondary axiswhen injected in ventral X. laevis blastomeres. On for-mation of a complex with a receptor of the frizzled family, �-catenin, the key signal transducer, is stabilizedand translocated into the cell nucleus (for review, seeGradl et al. 1999a and references therein). In the cyto-plasm, the phosphoprotein dishevelled is activated andinhibits glycogen synthase-kinase-3� (gsk-3�). Addi-tional proteins, which either inhibit or support gsk-3�

activity, bind to a multiprotein complex. The suppres-sion of gsk-3� is involved in dorsal signaling (He et al.1995; Yost et al. 1996). It prevents phosphorylation ofthe N-terminus of �-catenin, stabilizing it and allowingit to escape the ubiquitin–proteasome degradationpathway. In X. laevis, targets identified thus far are thehomeobox transcription factors siamois and twin andthe secreted protein nodal-related 3 (Xnr3), which isinvolved in neural differentiation and fibronectin syn-thesis (reviewed by Gradl et al. 1999a,b and referencestherein). Xtwin and siamois are transiently expressedin the dorsal marginal zone and the vegetal region fromthe mid-blastula to the gastrula stage (Laurent et al.1997).

The zebrafish bozozok gene locus encodes thedharma (dha) homeodomain protein. The activation ofboz/dha depends on �-catenin. A zygotic recessivebozozok mutant shows poor development of notochord,

prechordal mesoderm and forebrain (Driever et al.1997; Fekany et al. 1999). Some genes in the pre-sumptive dorsal mesoderm are not expressed. Thisshows that boz/dha is essential for only a part of dor-sal mesoderm differentiation. The homeodomain genevega1 inhibits the expression of boz/dha and causesventralization. Vega1 is activated at the mid-blastulatransition in all blastomeres, but is downregulated dor-sally before gastrulation. Ubiquitous expression ofvega1 is maintained in mutants in which boz/dha is dis-rupted, indicating a mutually antagonistic relationshipbetween the two genes (Kawahara et al. 2000b). TheN-terminal domain of boz/dha shares sequence simi-larity with a domain encoded by the X. laevis gsc gene,which acts as a transcriptional repressor (see earlier).In the embryo, vega1 may be eliminated by boz/dhain a limited region of the presumptive dorsal mesoderm.The expression of vega2 is initiated in a restricted ven-tral domain at the early gastrula stage. Together withvega1, it may negatively regulate organizer genes,including gsc, and participate in the fine regulation ofthe organizer region (Kawahara et al. 2000a). Thezebrafish vox and vent genes (Melby et al. 2000) sharesubstantial homology with the vega1 and vega2 genes,respectively.

The developmental importance of �-catenin was firstdemonstrated by its ability to induce an embryonic axis(Funayama et al. 1995). �-Catenin is associated withthe cytoplasmic domains of the plasma membranecadherin adhesion molecules. Overexpression of cad-herins as well as underexpression of �-catenin (by anantisense oligodeoxynucleotide complementary to �-catenin maternal mRNA) restrict the availability of �-catenin for the signaling pathway and inhibit dorsalmesoderm induction (Heasman et al. 1994). Depletionof �-catenin in animal caps of X. laevis embryos doesnot inhibit mesoderm induction by activin (Heasman etal. 1994). This shows that the activin- and Wnt-like �-catenin-pathways are separate (but not completelyindependent, see later) signaling pathways. �-Catenin,like Xwnt-8, is not a mesoderm inducer in animal caps.Disruption of the �-catenin gene in mice preventsmesoderm formation (Haegel et al. 1995).

�-Catenin is maternally expressed at the RNA andprotein levels. Zygotic transcription of �-catenin startsafter mid-blastula transition (Wylie et al. 1996). In X. lae-vis embryos, �-catenin displays greater cytoplasmicaccumulation along the entire future dorsal side by the2-cell stage. By the 16- to 32-cell stage, �-cateninaccumulates in dorsal, but not ventral, nuclei. �-Cateninis more stable at the dorsal side (Larabell et al. 1997).Experiments to show whether �-catenin is requiredbefore the mid-blastula transition have been inconclu-sive (Heasman et al. 1994; Wylie et al. 1996). However,


the injection into C4 (ventral) blastomeres of a gsc-luciferase reporter gene containing a response elementfor targets of the Wnt-8 �-catenin pathway, together witha Xwnt-8 cytoskeletal actin promoter construct, whichdirects the expression of Xwnt-8 only after the mid-blastula stage, does not activate the reporter gene. Thissuggests that the maternal �-catenin is active beforethe mid-blastula stage (Watabe et al. 1995). In the cellnucleus, �-catenin binds to high mobility group (HMG)box transcription factors of the TCF/LEF family, whichform a complex with DNA that displays an altered DNAbend (Grosschedl et al. 1994; Behrens et al. 1996).Chimeric factors constructed by fusion of �-catenindomains and the DNA binding domain of LEF 1 haveshown that the C-terminal transactivation domain of �-catenin is necessary and sufficient for signaling bythe LEF-1/�-catenin complex (Vleminckx et al. 1999).Based on these and other experiments, it has been pro-posed that the function of �-catenin in the nuclei is torecruit the transcription machinery to promoter regionsof the target genes (Hsu et al. 1998; Vleminckx et al.1999).

Sixteen different Xwnt molecules have so far beenidentified in X. laevis (reviewed by Gradl et al. 1999a);however, a Wnt-like factor in the dorsal �-catenin path-way (or another factor which causes the transfer of �-catenin from the cadherins to the cell nucleus) has yetto be identified. Loss-of-function experiments have notshown that Xwnt-8 or Xwnt-8a (in contrast to Xwnt-8 present in animal blastomeres) are required for endo-geneous axis specification in the intact X. laevisembryo (for review see Sokol 1996; Gradl et al. 1999a).

Fritz (frzb-1) encodes a protein related to the extra-cellular part of the frizzled Wnt transmembrane recep-tor and is an antagonist of Wnt signaling. Fritz can bindand inhibit Wnt-8 and is expressed in all three germlayers in early gastrulation (Leyns et al. 1997; Mayr et al. 1997; Wang et al. 1997). In addition to the frizzled-related proteins and the Dickkopf (Dkk) protein family,another Wnt-inhibitory factor, WIF-1, has been detectedin fish, amphibia and mammals. WIF-1 binds to Xwnt-8 in vitro. The gene is expressed in X. laevis embryosat the neurula stage. It is proposed to work togetherwith the other BMP inhibitors to fine-tune the spatial andtemporal pattern of Wnt activity (Hsieh et al. 1999).

Nodal and X. laevis nodal-related proteins andthe maternal transcription factor VegT

Nodal was first detected in mouse embryos. The nodalgene encodes a secreted TGF-�-type protein, whichis similar to the activin and BMP subgroups of the TGF-� superfamily. Unique to the nodal protein is a pair ofcysteine residues separated by two other amino acids(CXXC). Transcripts of the gene can be detected in the

primitive streak and the visceral endoderm of very earlystreak-stage embryos (Varlet et al. 1997). At gastrula-tion, they are localized in the anterior lip of the primi-tive streak surrounding the node (which is equivalentto the amphibian organizer). Mice homozygous for amutation of the nodal gene lack the primitive streak andmost of the mesoderm (Zhou et al. 1993; Coulon et al.1994). Nodal signaling is mediated by activin recep-tors and transcription factors of the Smad family.Member of the ECF-CFC family (homologs to zebrafishoep and mouse crypto) code for secreted factors thatregulate nodal signaling. It is possible that nodal actsonly in the presence of these proteins (reviewed bySchier & Shen 2000 and references therein).

Several nodal-related genes (Xnr1–6) are expressedin X. laevis embryos. Maternal Xnr or nodal transcriptscannot be detected. Xnr1 and Xnr2 transcripts appearfirst in vegetal cells of the late blastula. These genesmay be expressed in a dorsal to ventral gradient inendodermal cells (Agius et al. 2000). In the early gastrula, transcripts are found in the marginal zone, expression being most intense in the organizerregion (presumptive dorsal mesoderm). Xnr2 is alsoexpressed at low levels in pre-endodermal cells belowthe dorsal lip (Jones et al. 1995). Expression then disappears. Fugacin, a Xnr gene with the strongestsequence identity (53%) to mouse nodal, is expressedtransiently in the dorsal marginal zone (Ecochard et al.1998). Xnr4 is expressed preferentially in the endodermand in the dorsal mesoderm (Joseph & Melton 1997;Clements et al. 1999). Xnr5 and Xnr6 are induced aftermid-blastula transition in the vegetal region of theembryo, but not in the mesoderm (Takahashi et al.2000). Xnr1 expression reappears during the tail-budstages, initially near the posterior end of the notochordand then in a large asymmetric domain at the left lateral plate (Lustig et al. 1996a). The role of nodal sig-naling in left–right axis formation has been reviewedelsewhere (Schier & Shen 2000). Nodal-related genetranscriptional initiation sites used at the later stagesof development are different from those used at gas-trulation (Hyde & Old 2000).

Injection of Xnr1 and Xnr2 mRNA into X. laevisembryos results in substantial hyperdorsalization andincreased expression of gsc and muscle actin. In animal caps, they induce notochord and muscle(Jones et al. 1995). Xnr1 and Xnr2 can also induceendoderm (Yasuo & Lemaire 1999). The main nodal-related endodermal genes are Xnr5 and Xnr6.Besides endoderm, with the merkers endodermin andXsox 17b they induce notochord and muscle(Takahashi et al. 2000). The inducing activity of Xnr5 issimilar to the inducing activity of high doses of the vegetalizing factor (activin homolog; Asashima et al.1991c).


The regulation of Xnr genes is complex. Induction isa primary response, because de novo protein synthe-sis is not required. Xnr1 and Xnr2 can be induced inanimal ectoderm by activin (Jones et al. 1995), but Xnr5and Xnr6 cannot (Takahashi et al. 2000). Xnr genes arenot induced by bFGF. Xnr1 and Xnr2 are also inducedby an activin-like factor (processed Act-Vg1 construct)and by Xnr5 and Xnr6. The response is potentiated in�-catenin-injected explants, although �-catenin alonecannot activate Xnr1. Overexpression of VegT (amaternal transcription factor, see later) induces weakexpression of Xnr1 in animal caps, which is potentiatedby �-catenin (Agius et al. 2000). However, it is unlikelythat VegT alone can activate the expression of Xnr1 inthe vegetal region, because a reporter gene contain-ing VegT response elements is not coexpressed (Hyde & Old 2000). Xnr5 and Xnr6 can also not beinduced by �-catenin alone, but by a combination of �-catenin and VegT acting cooperatively (Takahashiet al. 2000).

The search for enhancer response elements in Xnrgenes is ongoing. Besides functional response ele-ments for VegT (Kofron et al. 1999; Hyde & Old 2000),a Fast site has been found in Xnr1 intron I, which iscompletely conserved between mouse nodal and Xnr1(Hyde & Old 2000). Mutational analysis has detectedtwo Wnt response elements in Xnr3 (McKendry et al.1997), which itself has no VegT response elements(McKendry et al. 1997; Hyde & Old 2000). These Wntresponse elements show striking homologies with ele-ments in Xnr1 (Hyde & Old 2000). In addition, Xnr1 canregulate its own expression by a positive regulatoryloop; it is probable that VegT can act together with �-catenin to induce Xnr1 expression (Hyde & Old 2000),Xnr1 can induce expression of VegT in ectodermal cells (Lustig et al. 1996b). In support of this, Derrière,another secreted protein of the TGF-� superfamily, canalso be regulated via VegT by a positive regulatory loop(Sun et al. 1999).

In the embryo, Xnr1 and Xnr2 mRNA are expressedin the marginal zone of early gastrulae, when the Apodprotein (expressed by VegT) is enhanced in this region(Stennard et al. 1999). The TGF-� signals for Xnr1 andXnr2 can be zygotic activin B (Dohrmann et al. 1993)and/or Xnr5 and Xnr6 (Takahashi et al. 2000). Toanswer this question, the spatial distribution and con-centration of the proteins must be known. Wnt/�-cateninsignaling cooperates with the other factors and a positive regulatory loop can enhance Xnr1 and Xnr2expression.

When the store of maternal VegT mRNA is depletedto 5–10% of the normal level by injection of an antisensedeoxyoligonucleotide into the vegetal pole of oocytes,the fate of the different regions of the blastula is com-pletely altered. The fate of the animal region is changed

from epidermis and nervous system to epidermis only;the fate of equatorial cells is changed from mesodermto ectoderm and ectoderm derivatives; and the vege-tal cell fate is changed from endoderm to mesodermand ectoderm. The marginal zone does not invaginate(Zhang et al. 1998). After injection of a very high doseof the antisense oligodeoxynucleotide, the embryos donot form a blastopore (Kofron et al. 1999). The doseused could have affected the specificity of theresponse. The low residual levels of mesodermalmarkers could be expressed by activin, the concen-tration of which is reduced, together with BMP-4, whichis not reduced.

Whether VegT acts cell-autonomously or by gener-ating signaling molecules has been investigatedthrough cell dissociation experiments in which cell tocell signaling is prevented (Clements et al. 1999). Thedata suggest that endodermal markers are cell-autonomously induced from the mid-blastula transitionon and that this induction must be rapidly reinforcedby signaling for expression to be maintained. In con-trast, mesodermal markers are entirely dependent on signaling.

Interesting results have been obtained by investi-gating the role of the VegT Antipodean (Apod) gene atthe level of the proteins, which were identified usingaffinity purified antibodies (Stennard et al. 1999).Western immunoblotting revealed two proteins of dif-ferent molecular weights, which could arise by alter-native splicing. The larger one, referred to as VegT (62 kDa), is expressed maternally. The protein is pre-sent from the egg to gastrulation in the presumptiveendoderm. The smaller one (60 kDa), referred to as Apod, is expressed only after the onset of zygotictranscription, initially in the presumptive endoderm andequatorial region, and only in the presumptive meso-dermal region from the early gastrula stage (stage 10.5)on. Apod is inducible by activin (Stennard et al.1999).The T-box of the T-box genes encodes a DNA-binding domain of �180 amino acids, which interactsas a dimer with the major and the minor grooves of theDNA (Mueller & Herrmann 1997).

The transcription factor XANF-1 is expressedthroughout the animal region and in the upper blasto-pore lip. Microinjection of XNAF-1 mRNA into animalblastomeres and the marginal zone leads to a massivemigration of cells to the interior of the embryo (Zaraiskyet al. 1995). The protein encoded by the Xrel gene, amember of the rel family of transcription factors, hasbeen detected in early cleavage stages and is dis-tributed in a gradient with the most intense staining inthe cytoplasm of the animal region to almost nothingin the vegetal region. Staining of the cell nuclei in theanimal cap and the marginal zone becomes apparentat the early blastula stage, shortly before the onset of


zygotic transcription (Bearer 1994). Loss-of-functionexperiments have so far not been carried out.

Cooperation of signaling pathways

The control of the activity of quite a number of genesduring embryogenesis depends on several distinct sig-naling pathways and nuclear factors. An example ofthe synergy of signaling pathways is the regulation ofthe siamois gene. Siamois can be induced in animalectoderm by the Wnt/�-catenin pathway, but not by the Smad1–BMP or Smad2–activin pathways. TheSmad2–activin pathway, however, can cooperate withthe �-catenin pathway to induce expression of siamoismore strongly than the �-catenin pathway alone. Thesignificance of this cooperation in normal embryos hasbeen shown by blocking the activin pathway with a trun-cated dominant-negative activin receptor. The expres-sion of siamois was three-fold reduced (Crease et al.1998). In another example, the Wnt/�-catenin pathwaycan enhance the induction of the dorsal mesodermalgsc (see also Watabe et al. 1995) and chordin genes(Crease et al. 1998). Experiments showing that the inhi-bition of Wnt-8 or BMP-4/2 signaling has a similar actionon the differentiation of mesoderm suggest coregula-tion by these factors (Hoppler & Moon 1998).

Xtwin is a target gene of �-catenin signaling medi-ated through the activity of the Lef1/Tcf proteins.Experiments with reporter gene constructs have shownthat Smad4 binding in proximity to the Lef1/Tcf bind-ing sites is needed for full activation. As described earlier, Smad4 is a mediator of BMP-2/4 as well asactivin signaling. Lef1 immunocoprecipitates withSmad4 in vitro. A model is proposed in which Smad4forms a complex with Lef1/Tcf and �-catenin to bind tothe Xtwin promoter region (Nishita et al. 2000).

In conclusion, mesoderm determination and differ-entiation could take place as a two-step process in thefollowing way. The dorsal mesoderm is determinedindependently of the endoderm by factors that arelocated in the marginal zone following cortical rotation.The factors responsible are probably BMP-2, activins(vegetalizing factor), FGF, eFGF and �-catenin, whichare all maternal proteins (Fig. 3). Receptors for BMP,activin and FGF are expressed maternally (or duringcleavage); �-catenin is located in cell nuclei along thewhole dorsal side at the cleavage stages. Mesodermdetermination could take place in a region where thesefactors overlap. A coarse pattern of receptor–effectorcomplexes could be formed. The mesodermal genesare in part immediate early genes, which can be tran-scribed without de novo protein synthesis. The ARF(see earlier) can form as early as the 8–16-cell stageand may be a component of the ‘molecular memory’,by which pre- and mid-blastula signals are maintaineduntil the onset of zygotic transcription (Huang et al.1995).

It is possible that mesodermal genes may beexpressed at a very low level prior to the mid-blastulastage, because RNA (Nakakura et al. 1987) and pro-tein (Tiedemann & Tiedemann 1954) are synthesizedin amphibian embryos at a very low level before themid-blastula transition. To show the expression of specific genes in cleavage and morula stages (withonly a low number of cells) would, however, requireextremely sensitive methods.

After mid-blastula transition, the nodal-related (Xnr)genes become very important for mesoderm and endo-derm differentiation and can, at least in part, replacethe activins and maintain and enhance mesoderminduction. Xnr1 and Xnr2 respond to activin, the vegetal Xnr5 and Xnr6 factors, Veg T and �-catenin

Fig. 3. Simplified scheme of the distribution of differentiation factors in amphhibian embryos.(A) Maternal factors in very earlycleavage to mid-blastula embryos.(B) Factors in the early gastrulastage. V, ventral; D, dorsal; AP, animal pole; VP, vegetal pole; BMP,bone morphogenetic protein; FGF,fibroblast growth factor; Xnr,Xenopus nodal-related protein.


signaling (Fig. 3). Proteins expressed by vegetal Xnrgenes could diffuse into the mesodermal marginalzone. Chordin, noggin, BMP-4, Xvents, FGF and Xwnt-8 are all involved in the development of the dorsoven-tral and posterior mesodermal pattern.

Determination of the vegetal endoderm before theearly gastrula stage is reversible, as is the case forectoderm (Wylie et al. 1987; see earlier). A ‘predeter-mination’ could occur via the presence of activin (veg-etalizing factor) in high concentrations, activin-relatedfactors, Wnt/�-catenin and the maternal VegT protein,which is still present in the presumptive endoderm afterthe onset of zygotic transcription until gastrulation(Stennard et al. 1999). This implicates VegT in the for-mation of the endoderm after mid-blastula transition,especially in the regulation of Xnr5 and Xnr6 expres-sion (Takahashi et al. 2000). VegT appears to beimportant for the equilibrium between endoderm andmesoderm. It prevents the extension of mesodermalgene expression beyond the presumptive endodermalregion of the embryo, but is involved, together withactivin, Xnr5 and Xnr6 and �-catenin, in the expressionof Xnr mesodermal genes. The ratio of the factors inthe different regions of the embryo will certainly beimportant. It is also likely that many factors involved inthe formation of the endomesodermal pattern have stillto be identified.

Preferential differentiation of single organs

Heart muscle, with its typical honeycomb-like appear-ance surrounded by an endothel-lined pericardial cavity, can, besides skeletal muscle and ventral meso-dermal tissues, be induced by recombinant bFGF athigh concentration in X. laevis ectoderm explants(Knöchel et al. 1989a). Beating hearts can also beinduced in 10–30% of explants by treatment of newtanimal caps with a high concentration (100 ng/mL for1 h) of activin, followed by sandwich culture withuntreated ectoderm for 2 weeks. Heartbeats are reg-ular and display the temperature-dependent frequencyof the normal newt heart (Ariizumi et al. 1996; Asashimaet al. 2000). The formation of heart tissue in embryosdepends on endoderm. Heart is not formed in T. alpestris when the endoderm is removed at the neuralplate stage (Mangold 1957). The deep dorsal endo-derm contributes to the specification of the heartanlage, whereas the superficial pharyngeal ectodermmay enhance heart morphogenesis during later stages(Sater & Jacobson 1989). The induction of endodermbesides mesoderm may be also a prerequisite for heartinduction. Grunz (1999a) observed a high percentageof heart-like structures and hearts when the upperblastoporal lip from early gastrulae of X. laevis, includ-

ing some head endomesoderm and ectoderm, wastreated with suramin (150 mM for 4 h) and cultured in vitro until normal larvae had reached the late neurula or tail-bud stages. These were then trans-planted into the posterior trunk area of host embryos,the heart anlage of which was exstirpated at the lateneurula stage. The host embryos could be rescued bythe transplant. Suramin is a polyanion that preventstranscription of dorsal marker genes in X. laevisembryos (Oschwald et al. 1993).

The Wnt antagonists Dkk1 and crescent, a frizzlerelated protein, can induce heart formation in the ven-tral posterior mesoderm of X. laevis explants. Inhibitionof BMP signaling does not prevent cardiogenesis.These and other results suggest that cardiogenesis isinduced in a region of high BMP and low Wnt-8a andWnt-3a activity (Maroon et al. 2001; Schneider &Mercola 2001).

Asashima and colleagues have induced a high fre-quency of pronephric tubules by treating X. laevis blas-tula ectoderm with 10 ng/mL recombinant activin and0.1 mM retinoic acid for 3 h. Retinoic acid alone had noinducing activity. When the induced explants weretransplanted into late neurula hosts, which were bilat-erally pronephrectomized, many of the hosts survivedup to 27 days. The pronephrectomized control embryosdeveloped edema and died after 5–9 days (Chan et al.1999).

The pancreas develops from the anterior endoderm(reviewed by Slack 1995). Contact of notochord withthe dorsal pancreatic bud is required for pancreaticdevelopment. The notochord represses endodermalShh in the dorsal pancreatic bud (Kim et al. 1997;Hebrok et al. 1998). Upper blastoporal lips of X. laevistreated with 0.1 mM retinoic acid for 3 h have beenshown to differentiate into pancreas-like structurestogether with notochord and endodermal epithelium.The pancreas-specific markers XIHbox 8 and insulinwere induced (Moriya et al. 2000a). Furthermore, iso-lated presumptive ectoderm from X. laevis blastulaewas treated with activin and retinoic acid to induce dif-ferentiation into pancreas (Moriya et al. 2000b). The dif-ferentiation of hematopoietic precursor cells, inducedby a low dose of activin (0.5 ng/mL) in X. laevis animalcap explants, to leukocyte and erythrocyte lineageswas promoted by the addition of murine stem cell factor and interleukin-11 (Miyanaga et al. 1998).

Concluding remarks

The isolation of pluripotent stem cells and novelinsights into embryonic differentiation provide new possibilities, especially to the field of transplantationmedicine. A thorough risk assessment must be carried


out for the use of stem cell therapy. It must be knownin stem cell therapy studies with the same certainty asin drug studies, which risks are involved and that noharm will occur to the patients. Using long-term experi-ments, the possibility that teratocarcinomas or tera-tomas will develop should be excluded. It has not beendifficult to foresee that ethical problems would arise(Tiedemann 1968). The use of human stem cellsderived from human embryos, which were only grownfor this purpose, has come under serious ethicalscrutiny. An alternative for some tissues would be theuse of somatic stem cells. Another possibility could bethe use of cell lines of robust and long-term prolifera-tion (EBD cells), which can be obtained after thera-peutic termination of pregnancy. Histocompatibilitycould be tested, as with other transplants, by tissue typ-ing, which can partially exclude an immunologicalrejection reaction. The most controversial question isthe so-called therapeutic cloning, which uses similarmethods as the cloning of almost identical animals. Thisis not far from human cloning, which is reminiscent ofthe nightmare of Aldous Huxley’s Brave New World (awarning to and criticism of the epoch) and which hasto be absolutely refused for ethical reasons and alsobecause of the malformations which occur. Therapeuticcloning would solve the problem of histocompatibility,but until now the frequency of mammalian nucleartransplants giving rise to blastocysts has been very low(�1/200). If this problem is solved (first of all in animalexperiments) and if public consent to the applicationof this method can be achieved, therapeutic cloningunder very strict legal conditions could be consideredas another alternative.

Our own responsibility rises with the new possibilitiesto interfere in human development. As Medawar (1984)expresses our obligation: ‘We must cultivate our garden’.

Acknowledgements

Our own investigations, reported in the present review,were supported by the Ministry of Education, Science,Sports and Culture of Japan, by the DeutscheForschungsgemeinschaft and by the Fonds derChemischen Industrie.

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