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Embryonic stem cells are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cell types, including germ cells. These qualities have made mouse embryonic stem cells a valuable resource for genetic manipulation of the mouse genome. In addition, they present a powerful system for the in vitro dissection of mammalian embryonic development. The recent isolation of human embryonic stem cells has raised a lot of interest for the potential of transposing our knowledge of lineage-specific differentiation of embryonic stem cells to cell-based therapy of human disease. Recent reports have provided insights into the specific differentiation of embryonic stem cells to different cell types of the embryo. However, progress in this direction seems to depend on the knowledge of the mechanisms controlling lineage decisions during embryogenesis.

Stem cells are functionally defined by their ability to self-renew and generate a progeny capable of creation or reconstitution of various tissues. Microarray analysis has shown a member of the transforming growth factor (TGF)-beta superfamily, Lefty, to be the single most abundant inhibitor in stem cells and in maternal decidua that supports embryo implantation. Lefty is regulated by pathways such as Smad (Sma and Mad [mothers against decapentaplegic]) and WNT (wingless-type) and by the transcriptional factor Oct3/4 (octamer-binding transcription factor 3/4), which support "stemness." Lefty is also induced upon exit from the state of stemness, including forced in vitro differentiation, and leukemia inhibitory factor withdrawal. Lefty is a candidate in cell-fate decisions because of its unique ability to modulate the expression of TGF-beta family proteins such as Nodal and by blanket inhibition of the activity of members of this family which require EGF-CFC (epidermal growth factor-Cripto, Frl-1, and Cryptic) as a coreceptor.

Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to derivatives of all three primary germ layers, yet little is known about the signaling cascades that govern their pluripotent character. Because it plays a prominent role in the early cell fate decisions of embryonic development, we have examined the role of TGFβ superfamily signaling in hESCs. We found that, in undifferentiated cells, the TGFβ/activin/nodal branch is activated (through the signal transducer SMAD2/3) while the BMP/GDF branch(SMAD1/5) is only active in isolated mitotic cells. Upon early differentiation, SMAD2/3 signaling is decreased while SMAD1/5 signaling is activated. We next tested the functional role of TGFβ/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extend these findings to show that SMAD2/3 activation is required downstream of WNT signaling, which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly, we show that in ex vivo mouse blastocyst cultures, SMAD2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a crucial role for TGFβ signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFβ and WNT signaling in these contexts.

Mammalian and Drosophila homologues of Baf57 have been previously isolated as being a subunit of SWI/SNF-like chromatin remodeling complexes. Here, we report the cloning and developmental expression of Xenopus Baf57. We isolated XBaf57 by using an expression cloning approach to identify novel modulators of Xenopus Smad7. XBaf57 co-operates with XSmad7 by increasing the expression of neural markers in ectodermal explants. XBaf57 is expressed in the ectoderm and pre-involuting mesoderm during gastrula stages and in the central nervous system during neurula and tailbud stages. These results raise the possibility that XBaf57 (or XBaf57-containing chromatin remodelling complexes) may be involved in the process of neural induction during Xenopus embryonic development.

Smad proteins are key intracellular signaling effectors for the transforming growth factor-β superfamily of peptide growth factors. Following receptor-induced activation, Smads move into the nucleus to activate transcription of a select set of target genes. The activity of Smad proteins must be tightly regulated to exert the biological effects of different ligands in a timely manner. Here, we report the identification of Smurf2, a new member of the Hect family of E3 ubiquitin ligases. Smurf2 selectively interacts with receptor-regulated Smads and preferentially targets Smad1 for ubiquitination and proteasome-mediated degradation. At higher expression levels, Smurf2 also decreases the protein levels of Smad2, but not Smad3. In Xenopus embryos, ectopic Smurf2 expression specifically inhibits Smad1 responses and thereby affects embryonic patterning by bone morphogenetic protein signals. These findings suggest that Smurf2 may regulate the competence of a cell to respond to transforming growth factor-β/bone morphogenetic protein signaling through a distinct degradation pathway that is similar to, yet independent of, Smurf1.

In Xenopus, endodermal cell fate is determined gradually from late blastula to early gastrula stages; cell-cell interaction plays an important role in this process. Here we use a cell dissociation assay to show that extracellular signaling is required continuously before endoderm determination. Activin and Vg1, but not BMP2 or basic FGF, rescue the expression of endodermal markers in dissociated cells when provided at the mid-blastula transition (MBT, the time in which zygotic transcription begins). Removal of exogenously added activin or Vg1 before MBT results in reduction of endodermal gene expression in dissociated vegetal cells. In vivo, endogenous endodermal markers are reduced in vegetal explants when activin-like signaling is blocked with dominant negative receptors. VegT, a maternal transcription factor shown to be critical for endoderm specification, relies on an active TGFbeta pathway to induce endoderm in animal caps. These results indicate that TGFbeta signaling may be activated by the maternally expressed VegT to participate in endoderm determination. In addition, VegT function seems to be required in parallel with the TGFbeta pathway, as overexpression of activin does not relieve endoderm repression by a dominant negative VegT mutant in vegetal cells. Our data suggest that maternal VegT first activates a zygotic TGFbeta signal, then cooperates with this signal to determine the endodermal cell fate.

Screening a maternal Xenopus expression library for activities that synergize with low levels of injected β -catenin, we have isolated a clone encoding the C-terminal end of x- β TrCP-2, a highly conserved protein belonging to the F-box/WD40 family of ubiquitin-ligase specificity factors. We show that x- β TrCP-2 expression reduces dorsal axis formation in Xenopus embryos. A dominant negative mutant lacking the F-box triggers the opposite effect, inducing secondary axes and activating the expression of Wnt responsive genes in ectodermal explants. In light of the existence of β TrCP transcripts associated with the vegetal cortex, we propose that β TrCP plays a fundamental role in the establishment of the dorsal determinants during cortical rotation in Xenopus .

During embryogenesis, inductive interactions underlie the development of much of the body plan. In Xenopus laevis, factors secreted from the vegetal pole induce mesoderm in the adjacent marginal zone; members of both the transforming growth factor-beta (TGF-beta) and fibroblast growth factor (FGF) ligand families seem to have critical roles in this process. Here we report the identification and characterization of laloo, a novel participant in the signal transduction cascade linking extracellular, mesoderm-inducing signals to the nucleus, where alteration of cell fate is driven by changes in gene expression. Overexpression of laloo, a member of the Src-related gene family, in Xenopus embryos gives rise to ectopic posterior structures that frequently contain axial tissue. Laloo induces mesoderm in Xenopus ectodermal explants; this induction is blocked by reagents that disrupt the FGF signalling pathway. Conversely, expression of a dominant-inhibitory Laloo mutant blocks mesoderm induction by FGF and causes severe posterior truncations in vivo. This work provides the first evidence that a Src-related kinase is involved in vertebrate mesoderm induction.

During gastrulation in vertebrates the cells of the embryonic ectoderm give rise to epidermal progenitors in the ventral side and neural progenitors in the dorsal side. Despite many years of scrutiny, the molecular basis of these important embryonic cell fate decisions have not been solved. Only recently have we witnessed swift progress in the quest for factors involved in neural and epidermal induction. Several of what seem to be bona fide in vivo neural and epidermal inducers have been cloned, and the mechanism of their functions in embryos is also beginning to be understood. These new molecular results have revolutionized our view on the patterning of embryonic ectoderm and suggest that while the induction of epidermis requires instructive inductive signals, the establishment of neural fate occurs by default when epidermal inducers are inhibited. In this review, we discuss recent advances of our knowledge on epidermal and neural induction in the context of the “default model”. We will then address the process of neurogenesis as well as recent findings on neural patterning. Emphasis is placed on, but not limited to, discoveries made in Xenopus, as most of our progress in understanding the ectodermal patterning is obtained from studies using this organism. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 128–151, 1998

We have identified a short segment of the mouse Pax-6 gene 5' flanking region that is necessary and sufficient for reporter construct expression in components of the eye derived from non-neural ectoderm. This transcriptional control element has a highly conserved nucleotide sequence over 341 bp and is located approximately 3.5 kb upstream of the start-point for transcription from the most proximal promoter (PO) of the Pax-6 gene. The level of identity between the human and mouse Pax-6 genes in this region is 93%. When combined either with its natural promoter or a heterologous minimal promoter, this element directs reporter construct expression to a region of surface ectoderm overlying the optic cup beginning at E8.5-9.0 (12-14 somites). Subsequently, expression is restricted to the lens (primarily the lens epithelium) and the corneal epithelium. This element will provide an important tool in future transgenic analyses of lens formation and will allow identification of transcription factors with a central function in lens development.

The specification of neural fate in Xenopus embryos has been shown to be under regulation by negative factors. The secreted protein bone morphogenetic protein-4 (BMP4) has been identified as one of these factors: in the early gastrula ectoderm, BMP4 can both inhibit neural fate and induce epidermis. In this study, we show that two other Xenopus BMP genes, BMP2 and BMP7, are endowed with the same types of activities. First, we show that expression of a dominant negative form of the BMP2 ligand, which blocks normal processing of the wild-type ligand, causes neuralization of Xenopus ectoderm. Second, we have isolated the Xenopus BMP2/7 receptor (XALK2) and generated a constitutively active mutant that signals in a ligand-independent manner. We show that signals from the activated BMP2/7 receptor also inhibit neuralization and induce epidermis in dissociated ectoderm cells. Consistent with both findings we show that secreted BMP2 and BMP7 ligands can also mediate neural inhibition and epidermal induction. These results suggest that both BMP2 and BMP7 may be involved independently or together with BMP4 in the inhibition of the neural fate and the onset of the epidermal induction pathway in vivo. This further supports the idea that epidermal induction is due to the effects of multiple signals from heterogeneous BMP genes.

Epidermal fate in Xenopus ectoderm has been shown to be induced by a secreted growth factor, Bone Morphogenetic Protein 4 (BMP4). However, the molecular mechanism mediating this response is poorly understood. Here, we show that the expression of the homeobox gene, msx1, is an immediate early response to BMP4 in Xenopus embryos. The timing of expression and embryonic distribution of msx1 parallel those described for BMP4. Moreover, over-expression of msx1 in early Xenopus embryos leads to their ventralization as described for BMP4. Consistent with mediating a BMP type of signaling, overexpression of msx1 is sufficient to induce epidermis in dissociated ectoderm cells, which would otherwise form neural tissue. Finally, msx1 can also rescue neuralization imposed by a dominant negative BMP receptor (tBR) in ectodermal explants. We propose that Xenopus msx1 acts as a mediator of BMP signaling in epidermal induction and inhibition of neural differentiation.

Morphogens are thought to establish pattern in early embryos by specifying several cell fates along a gradient of concentration; a well-studied example is the Drosophila protein decapentaplegic (DPP) acting in the wing disc. Recent work has established that bone morphogenetic protein 4 (BMP4), the vertebrate homologue of DPP, controls the fundamental choice between neural and epidermal fates in the vertebrate ectoderm, under the control of antagonists secreted by the organizer region of the mesoderm. We now show that BMP4 can act as a morphogen, evoking distinct responses in Xenopus ecto-dermal cells at high and low concentrations, in a pattern consistent with the positions of the corresponding cell types in the embryo. Moreover, this complex cellular response to extracellular BMP4 concentration does not require subsequent cell-cell communication and is thus direct, as required of a classical morphogen. We also show that the same series of cell types – epidermis, cement gland and neural tissue – can be produced by progressively inhibiting endogenous BMP signaling with specific antagonists, including the organizer factor noggin. Finally, expression of increasing doses of the signal transduction molecule Smad1 accurately reproduces the response to BMP4 protein. Since Smads have been shown to act in the nucleus, this finding implies a direct translation of extracellular morphogen concentration into transcription factor activity. We propose that a graded distribution of BMP activity controls the specification of several cell types in the gastrula ectoderm and that this extracellular gradient acts by establishing an intracellular and then nuclear gradient of Smad activity.

The Smad proteins have been implicated in the intracellular signaling of transforming growth factor-β (TGF-β) ligands. Here we describe the function of Smad5 in early Xenopus development. Misexpression of Smad5 in the embryo causes ventralization and induces ventral mesoderm. Moreover, Smad5 induces epidermis in dissociated ectoderm cells which would otherwise form neural tissue. Both of these activities require Smad4 (DPC4) activity. We propose that Smad5 acts downstream of the BMP4 signaling pathway in Xenopus embryos and directs the formation of ventral mesoderm and epidermis.

Activins and other ligands in the TGFb superfamily signal through a heteromeric complex of receptors. Disruption of signaling by a truncated type II activin receptor, XActRIIB (previously called XAR1), blocks mesoderm induction and promotes neuralization in Xenopus embryos. We report the cloning and characterization of a type I activin receptor, XALK4. Like truncated XActRIIB, a truncated mutant (tXALK4) blocks mesoderm formation both in vitro and in vivo; moreover, an active form of the receptor induces mesoderm in a ligand-independent manner. Unlike truncated XActRIIB, however, tXALK4 does not induce neural tissue. This difference is explained by the finding that tXALK4 does not block BMP4-mediated epidermal specification, while truncated XActRIIB inhibits all BMP4 responses in embryonic explants. Thus, the type I and type II activin receptors are involved in overlapping but distinct sets of embryonic signaling events.

Although noggin, follistatin, and chordin can neuralize by antagonizing BMP4 epidermalizing activity, is it possible that, in addition, they transduce a signal via a receptor, as was originally postulated for noggin and chordin? The default model would predict not. There are indeed three lines of evidence that strongly argue against the existence of receptors for follistatin, noggin, or chordin, at least in the pathway mediating neuralization, and that inhibition of BMP signaling is sufficient to unveil the neural fate. First, there is the evidence from cell dissociation experiments discussed above: when embryonic cells are dissociated for several hours they will make neural tissue (3xNeural differentation of Xenopus laevis ectoderm takes place after disaggregation and delayed reaggregation without inducer. Grunz, H and Tacke, L. Cell Diff. Dev. 1989; 28: 211–218Crossref | PubMed | Scopus (157)See all References, 2xSingle cell analysis of mesoderm formation in the Xenopus embryo. Godsave, S.F and Slack, J.M.W. Development. 1991; 111: 523–530PubMedSee all References), and the addition of BMP4 will inhibit this effect and induce epidermis (Wilson and Hemmati-Brivanlou 1995xInduction of epidermis and inhibition of neural fate by Bmp-4. Wilson, P.A and Hemmati-Brivanlou, A. Nature. 1995; 376: 331–333Crossref | PubMedSee all ReferencesWilson and Hemmati-Brivanlou 1995). The second evidence comes from experiments recently performed in Drosophila. Holley et al. 1996xThe Xenopus dorsalizing factor noggin ventralizes Drosophila embryos by preventing DPP from activating its receptor. Holley, S, Neul, J, Attisano, L, Wrana, J, Sasai, Y, O'Connor, M, De Robertis, E, and Ferguson, E. Cell. 1996; 86: 607–617Abstract | Full Text | Full Text PDF | PubMed | Scopus (180)See all ReferencesHolley et al. 1996 demonstrate that at least for SOG/chd, and perhaps for noggin, binding DPP/BMP is their only function. They showed that while noggin inhibits DPP and phenocopies a dpp− mutation, it can only operate outside of the cell, and in the presence of an activated DPP receptor, its effect is abolished. More compelling is the fact that the double sog− dpp− mutant has the same phenotype as the dpp− mutant. If SOG had any other function than just inhibiting DPP, the double mutant phenotype should have been different than that of dpp− alone. The final line of evidence comes from the fact that, while chordin can reverse the osteogenic induction caused by BMP4 in 10T1/2 cell lines, it cannot block the one mediated by retinoic acid (Piccolo et al. 1996xDorsoventral patterning in Xenopus (inhibition of ventral signals by direct binding of chordin to BMP-4) . Piccolo, S, Sasai, Y, Lu, B, and De Robertis, E. Cell. 1996; 86: 589–598Abstract | Full Text | Full Text PDF | PubMed | Scopus (797)See all ReferencesPiccolo et al. 1996). Taken together, these observations strongly suggest that inhibition of BMP signaling is sufficient for neuralization.Contribution from many groups working with the amphibian system has provided a molecular solution to the problem of vertebrate neural induction originally defined by Spemann and Mangold. The challenge for the future will inevitably include the establishment of a link between the early neural specification process, described above, and the function of neurogenic genes operating downstream of these signaling events, ultimately leading to the generation of a mature neuron.

An early milestone in the development of the vertebrate central nervous system (CNS) occurs soon after gastrulation when the neural plate forms on the dorsal surface of the embryo. Appearance of the neural plate marks the generation of committed neural progenitors that proliferate and eventually gives rise to the cell types comprising the CNS. The importance of determining how a particular region of the embryo forms the neural plate led to the discovery of neural induction in the early part of the last century. We will first describe how the process of embryonic induction was defined experimentally, how the mechanisms involved are evolutionarily conserved, and how the molecular circuitry that underlies this process was unraveled more recently. In addition, as the neural plate forms, the cells within it are molecularly distinct according to their position along the body axis. Regionalization of the neural plate is the first step in neural patterning, the events that allow progenitors to form different types of neural tissue along the body axis. We describe how regionalization of the nervous system begins at the neural plate stage, focusing on how polarity along the anteroposterior axis is first established. Finally, understanding the mechanisms that allow neural tissue to form in the embryo and become regionalized has been influential in more recent attempts to guide the differentiation of embryonic stem cells (ESCs) in culture. We will describe how the mechanisms that determine early cell fate in embryos have been used to devise protocols for guiding ESC differentiation.

In Xenopus embryos growth cones of the mandibular trigeminal nerve contact cells located in the posterior end of the cement gland. We report three lines of evidence suggesting that cues from the posterior cells of the target influence the behavior of trigeminal neurites. First, target ablation in vivo results in failure of axons to stop and to arborize. Second, 180 degree rotation in the anteroposterior axis of the target results in anterior innervation of the cement gland. Third, ectopic cement gland implantation in the path of neurites migration leads to ectopic innervation. These results provide evidence for short-range cues (50 microns) in the posterior part of the cement gland controlling the stopping and the branching of the mandibular trigeminal nerve. Furthermore, we show that an ectodermic explant expressing follistatin mimics the natural target in homing trigeminal neurites. We finally propose to use this novel in vivo assay to isolate the trigeminal nerve target-recognition molecule(s).

We have analyzed two gene products expressed in the early endoderm of Xenopus laevis: Xlhbox-8, a pancreas-specific transcription factor and intestinal fatty acid binding protein (IFABP), a marker of small intestinal epithelium. Expression of the pancreas marker relies on cell signaling mediated by both the TGF-beta and FGF classes of secreted peptide growth factors, whereas, expression of the more posterior small intestinal marker does not. Endodermal explants devoid of mesoderm express both markers in a regionalized manner. Cortical rotation is required for the expression of the more anterior marker, Xlhbox-8, but not for the small intestinal marker, IFABP. These findings suggest that endodermal patterning is dependent, in part, on the same events and signals known to play important roles in mesodermal development. Furthermore, inhibition of TGF-beta signaling in the endoderm leads to ectopic expression of both mesodermal and ectodermal markers, suggesting the TGF-beta signaling may play a general role in the segregation of the three embryonic germ layers.

In order to study anteroposterior neural patterning in Xenopus embryos, we have developed a novel assay using explants and tissue recombinants of early neural plate. We show, by using region-specific neural markers and lineage tracing, that posterior axial tissue induces midbrain and hindbrain fates from prospective forebrain. The growth factor bFGF mimics the effect of the posterior dorsal explant in that it (i) induces forebrain to express hindbrain markers, (ii) induces prospective hindbrain explants to make spinal cord, but not forebrain and midbrain, and (iii) induces posterior neural fate in ectodermal explants neu-ralized by the dominant negative activin receptor and follistatin without mesoderm induction. The competence of forebrain explants to respond to both posterior axial explants and bFGF is lost by neural groove stages. These findings demonstrate that posterior neural fate can be derived from anterior neural tissue, and identify a novel activity for the growth factor bFGF in neural patterning. Our observations suggest that full anteroposterior neural patterning may be achieved by caudalization of prospec-tive anterior neural fate in the vertebrate embryo.

We provide a comparative analysis of the expression patterns and ventral mesoderm-inducing properties of Xenopus BMP-2 and BMP-4. Transcripts for BMP-2 and BMP-4 are maternally stored in eggs, and zygotic expression of these genes is uniform in the ectoderm and mesoderm in late blastulae. During gastrulation, BMP-2 is expressed at a low level throughout the ectoderm and marginal zone, but at early neurula stages a patch of dorso-anterior cells displays enhanced expression. In contrast, BMP-4 transcripts are restricted to the ventrolateral marginal zone during gastrulation, and in late gastrula and early neurula BMP-4 is expressed in the epidermis but not the neural plate. At post-neurula stages, BMP-2 and BMP-4 transcripts are associated with a variety of mesodermal structures, including the pharyngeal pouches, heart, blood island, and blastopore. At tailbud stages, BMP-2 and BMP-4 are expressed in neural tissues including the neural tube and brain. In mesoderm induction assays, BMP-2 and BMP-4 induce Xhox3, an early ventral-posterior mesoderm marker, and larval βT1 globin, a marker for red blood cells. Induction of red blood cells in response to BMP-4 was demonstrated by staining with a hemoglobin-specific reagent. Little is known about factors that induce hematopoietic lineages in vertebrates, and these results provide evidence linking BMP activity and blood differentiation. Globin induction by BMP-2 and BMP-4 is blocked by co-expression of a dominant-negative activin receptor, suggesting that either endogenous activin signals are required for BMP-mediated induction, or that the trancated activin receptor interferes with signaling by BMP receptors. In assays on marginal zone explants, we demonstrate that BMP-4 respecifies dorsal mesoderm to form ventral mesoderm, consistent with its ability to induce blood and to ventralize embryos. BMP-2, however, does not display such activity. The findings extend and support evidence that BMP-2 and BMP-4 function in ventral mesoderm induction and patterning in Xenopus. Our data furthermore high light the multiple functions these factors fulfill during early vertebrate embryogenesis. © 1995 Wiley-Liss, Inc.

Activins are members of the transforming growth factor-beta superfamily, a class of peptide growth factors that can regulate the growth and differentiation of a variety of cell types. In mesoderm induction assays, activins A and B were shown to be very potent inducers and it was only recently demonstrated that they are crucial for initial mesoderm induction in Xenopus embryos. To determine the source of activin protein for initial mesoderm induction and to investigate whether activins may play further roles in embryonic development we have examined the localization of the mRNAs encoding the activin beta A and beta B subunits in Xenopus embryos. Activin beta A and beta B mRNAs are found in the follicle cells surrounding oocytes but not in oocytes themselves or fertilized eggs. During embryogenesis activin mRNA is first detected after the midblastula transition and expression increases as development proceeds. Activin beta B mRNA is homogeneously distributed during blastula and early gastrula stages but restricted to the dorso-anterior region in neurula stage embryos. At the early tailbud stage activin expression becomes confined to the brain, eye analgen, visceral pouches, otic vesicles, and the anterior notochord.

In Xenopus embryos, a truncated type II activin receptor (Delta1XAR1), capable of blocking signals by several transforming growth factor (TGF)-beta family members, can induce neural tissue suggesting neural fate is under inhibitory control. Activin and bone morphogenetic protein 4 (BMP4) can act as neural inhibitors but only BMP4 can induce epidermis in Xenopus ectodermal cells. We have used the pluripotent mouse embryonal carcinoma cell line P19 to examine whether the mechanisms of ectodermal cell fate decisions are conserved among vertebrates. We show that a P19 cell line expressing Delta1XAR1 will differentiate into neurons. In addition, BMP4 inhibits retinoic acid (RA)-induced neural differentiation of P19 cells and induces keratin expression. These results suggest that in mammals as in amphibians neural fate is under inhibitory control and BMP4 can alter ectodermal differentiation.