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Abstract Background Microarray studies of the same phenomenon in different labs often appear at variance because the published lists of regulated transcripts have disproportionately small intersections. We demonstrate that comparing studies by intersecting lists in this manner is methodologically flawed by reanalyzing three studies of the molecular signature of "stemness" in human embryonic stem cells. There are only 7 genes common to all three published lists, suggesting disagreement. Results Carefully reanalyzing all three together from the raw data we detect 111 genes upregulated and 95 downregulated in all three studies. The upregulated list was subject to rtRTPCR analysis and 75% of the genes were confirmed. Conclusion Our findings show that the three studies have a substantial core of common genes, which is missed if only the published lists are examined. Combined analysis of multiple experiments can be a powerful way to distil coherent conclusions.

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. [Copyright &y& Elsevier]

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)-β 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-β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Microarray studies of the same phenomenon in different labs often appear at variance because the published lists of regulated transcripts have disproportionately small intersections. We demonstrate that comparing studies by intersecting lists in this manner is methodologically flawed by reanalyzing three studies of the molecular signature of "stemness" in human embryonic stem cells. There are only 7 genes common to all three published lists, suggesting disagreement. Results: Carefully reanalyzing all three together from the raw data we detect 111 genes upregulated and 95 downregulated in all three studies. The upregulated list was subject to rtRTPCR analysis and 75% of the genes were confirmed. Conclusion: Our findings show that the three studies have a substantial core of common genes, which is missed if only the published lists are examined. Combined analysis of multiple experiments can be a powerful way to distil coherent conclusions. [ABSTRACT FROM AUTHOR]

Reports on the identification of Smurftwo, a member of the Hect family of ubiquitin ligases. Selective interaction of Smurftwo with receptor-regulated Smads; Preferential targeting of Smadone for ubiquitination and proteasome-mediated degradation; Effect of Smurftwo expression on embryonic patterning by bone morphogenetic protein signals.

The formation of the vertebrate nervous system is initiated at gastrula stages of development, when signals from a specialized cluster of cells (the organizer) trigger neural development in the ectoderm. This process, termed neural induction, was first described in 1924 and stemmed from experiments on amphibia (Spemann & Mangold 1924). In recent years, the molecular mechanisms underlying neural induction in the amphibian have been elucidated. Surprisingly, neuralizing agents secreted by the organizer do not act via receptor-mediated signaling events; rather, these factors antagonize local epidermal inducers within the cells of the dorsal ectoderm and function to uncover the latent neural fate of these cells. Many of the recent advances in our understanding of vertebrate neural induction come from studies on the frog, Xenopus laevis. It is now clear that a blockade of signaling of the bone morphogenetic proteins (BMPs) during gastrula stages is sufficient to initiate neuralization of the ectoderm in this species. Thus this review first details our current understanding of neural induction, using the amphibian as a model. We then use data emerging from other systems to examine the extent to which the Xenopus studies can be applied to other vertebrate species. The initiation of the neurectoderm-specific gene expression program and subsequent steps in patterning and neuronal development are only touched on here. We focus primarily on the initial establishment of the neural fate in the vertebrate gastrula ectoderm. [ABSTRACT FROM AUTHOR]

Provides information on a study which examined the factors involved in the specification of neural fate within the frame of the default model of neural introduction in vertebrates. Lessons from classical experimental embryology; Molecular approaches and the notion of the default state; Evidence in favor of the default model of neurogenesis; Competence of the ectoderm and the default model; Conclusion.

Investigates the in vivo role of activins in mesoderm induction. Identification of two maternally expressed `Xenopus' activin receptors; Mesoderm induction in explants blocked by truncated activin receptor (tAR); Expression of ectoderm-specific genes; Endogenous mesoderm inducing signal inhibited; Phenotype expressing tAR in whole embryos; Rescue by wild-type activin receptor; Discussion.

Studies neural induction and its effects to the embryogenesis of the vertebrate nervous system. Occurrence of neutralization of embryonic cells; Development of the concept of neural induction; Gene products responsible for neural induction.

Studies the expression of the activin antagonist follistatin. Neuralization of ectodermal explants; Localization of follistatin to Spemann organizer; Cloning of follistatin from Xenopus embryos.

Studies the role of inhibition of activin receptor signaling in neuralization of Xenopus. Expression of a truncated activin type II receptor; Identification of molecules involve in formation of vertebrate nervous system; Diversion of prospective ectodermal and endodermal blastomeres to a neural fate.

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. [Copyright &y& Elsevier]

Using an expression cloning approach, we have unveiled a novel function for the transcription factor E2F. We demonstrate that Xenopus E2F (xE2F) is required for patterning of the Xenopus embryonic axis. Overexpression of xE2F in embryos induces ectopic expression of ventral and posterior markers, including selected members of the Hox genes, and suppresses the development of dorsoanterior structures. Loss of xE2F function in embryos leads to the elimination of ventral and posterior structures. These observations suggest that xE2F acts as an important regulator of region-specific gene expression and in the formation of the embryonic axis. This study provides evidence for an additional embryonic function for E2F, independent of its well-documented role in cell cycle regulation, and suggests a novel mechanism of region-specific gene expression during vertebrate embryogenesis.

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.

We have identified the 30-zinc finger protein OAZ as a DNA-binding factor that associates with Smads in response to BMP2, forming a complex that transcriptionally activates the homeobox regulator of Xenopus mesoderm and neural development, Xvent-2. OAZ contains a BMP signaling module formed by two clusters of fingers that bind Smads and the Xvent-2 BMP response element, respectively. Previously implicated as a transcriptional partner of Olf-1/EBF in olfactory epithelium and lymphocyte development in the rat, OAZ fulfills this role through clusters of fingers that are separate from the BMP signaling module. The mutually exclusive use of OAZ by the BMP-Smad and Olf pathways illustrates the dual role of a multi-zinc finger protein in signal transduction during development.

We report here that misexpression of the transcription factor Pax6 in the vertebrate Xenopus laevis leads to the formation of differentiated ectopic eyes. Multiple molecular markers indicated the presence of mature lens fiber cells, ganglion cells, Müller cells, photoreceptors and retinal pigment epithelial cells in a spatial arrangement similar to that of endogenous eyes. Lineage tracing experiments showed that lens, retina and retinal pigment epithelium arose as a consequence of the cell-autonomous function of Pax6. These experiments also reveal that the cell autonomous activity of misexpressed Pax6 causes the ectopic expression of a number of genes including Rx, Otx2, Six3 and endogenous Pax6, each of which has been implicated in eye development. The formation of ectopic and endogenous eyes could be suppressed by coexpression of a dominant-negative form of Pax6. These data show that in vertebrates, as in the invertebrate Drosophila melanogaster, Pax6 is both necessary and sufficient to trigger the cascade of events required for eye formation.

In Xenopus, ectodermal cell fates are determined by antagonistic interaction between the BMP subfamily of TGF-(beta) ligands and the organizer-specific secreted factors (e.g. noggin, chordin and follistatin). Inhibition of BMP function by these factors can convert cells from an epidermal to a neural cell fate. In this study, we report that GDF6, a new member of the Xenopus TGF-(beta) family, can function in antagonistic interaction with neural inducers. GDF6 induces epidermis and inhibits neural tissue in dissociated cells, and this activity is blocked by the presence of noggin. We demonstrate that GDF6 binds directly to the neural inducer noggin. Furthermore, we find that GDF6 and BMP2 can form heterodimers and the process seems to require cotranslation of the proteins in the same cells. In normal embryos, GDF6 and BMP2 are coexpressed in several places, including the edge of the neural plate at early neurula stages, suggesting that GDF6 may synergize with BMPs to regulate patterning of the ectoderm. Our data show for the first time that noggin can bind directly to and inhibit another TGF-(beta) family member: GDF6. In addition, BMP and GDF6 heterodimers may play an important role in vivo to regulate cell fate determination and patterning.

Screening a maternal Xenopus expression library for activities that synergize with low levels of injected beta-catenin, we have isolated a clone encoding the C-terminal end of x-beta TrCP-2, a highly conserved protein belonging to the F-box/WD40 family of ubiquitin-ligase specificity factors. We show that x-beta 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 beta TrCP transcripts associated with the vegetal cortex, we propose that beta TrCP plays a fundamental role in the establishment of the dorsal determinants during cortical rotation in Xenopus.

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., (Copyright 1998 Elsevier Science Ireland Ltd. All rights reserved.)

Goosecoid (Gsc) is a homeodomain protein expressed in the organizer region of vertebrate embryos. Its Drosophila homologue, D-Gsc, has been implicated in the formation of the Stomatogastric Nervous System. Although there are no apparent similarities between the phenotypes of mutations in the gsc gene in flies and mice, all known Gsc proteins can rescue dorsoanterior structures in ventralized Xenopus embryos. We describe how D-Gsc behaves as a transcriptional repressor in Drosophila cells, acting through specific palindromic HD binding sites (P3K). D-Gsc is a 'passive repressor' of activator homeoproteins binding to the same sites and an 'active repressor' of activators binding to distinct sites. In addition, D-Gsc is able to strongly repress transcription activated by Paired-class homeoproteins through P3K, via specific protein-protein interactions in what we define as 'interactive repression'. This form of repression requires the short conserved GEH/eh-1 domain, also present in the Engrailed repressor. Although the GEH/eh-1 domain is necessary for rescue of UV-ventralized Xenopus embryos, it is dispensable for ectopic induction of Xlim-1 expression, demonstrating that this domain is not required for all Gsc functions in vivo. Interactive repression may represent specific interactions among Prd-class homeoproteins, several of which act early during development of invertebrate and vertebrate embryos.

Neural patterning occurs soon after neural induction during early development. In Xenopus, several caudalizing factors transform anterior neural to posterior neural tissue at the open neural plate stages, while other factors are responsible for setting up mediolateral polarity which becomes the dorsoventral (D-V) axis after neural tube closure. Many Wnt ligands are expressed in the neural tube in distinct anteroposterior (A-P) and D-V domains, implying a function in neural patterning. Here we report the cloning of a full-length Xenopus Wnt7B gene. Xwnt7B induces neural crest markers Xslug and Xtwist in ectodermal explants coinjected with neural inducer noggin and in ectodermal cells neuralized by dissociation. In vivo, Xwnt7B expands the Xtwist expression domain when injected in the animal pole. Our results suggest that Wnt members are involved in dorsoventral patterning of the neural tube., (Copyright 1998 Academic Press.)

Bone morphogenetic protein (BMP) receptors signal by phosphorylating Smad1, which then associates with Smad4; this complex moves into the nucleus and activates transcription. Here we report the existence of a natural inhibitor of this process, Smad6, a longer version of the previously reported JV15-1. In Xenopus embryos and in mammalian cells, Smad6 specifically blocks signaling by the BMP/Smad1 pathway. Smad6 inhibits BMP/Smad1 signaling without interfering with receptor-mediated phosphorylation of Smad1. Smad6 specifically competes with Smad4 for binding to receptor-activated Smad1, yielding an apparently inactive Smad1-Smad6 complex. Therefore, Smad6 selectively antagonizes BMP-activated Smad1 by acting as a Smad4 decoy.

Netrin-1 promotes outgrowth of axons in vitro through the receptor Deleted in Colorectal Cancer (DCC) and elicits turning of axons within embryonic explants when presented as a point source. It is not known whether netrin-1 alone can elicit turning nor whether DCC mediates the turning response. We show that Xenopus retinal ganglion cell growth cones orient rapidly toward a pipette ejecting netrin-1, an effect blocked by antibodies to DCC. In vitro, netrin-1 induces a complex growth cone morphology reminiscent of that at the optic nerve head, a site of netrin-1 expression in vivo. These results demonstrate that netrin-1 can function alone to induce turning, implicate DCC in this response, and support the idea that netrin-1 contributes to steering axons out of the retina.

Bone Morphogenetic Protein-4 (BMP-4) is a potent epidermal inducer and inhibitor of neural fate. We have used differential screening to identify genes involved in epidermal induction downstream of BMP-4 and report here evidence of a novel translational mechanism that regulates the division of the vertebrate ectoderm into regions of neural and epidermal fate. In dissociated Xenopus ectoderm, addition of ectopic BMP-4 leads to an increase in the expression of translation initiation factor 4AIII (eIF-4AIII), a divergent member of the eIF-4A gene family until now characterized only in plants. In the gastrula embryo, Xenopus eIF-4AIII (XeIF-4AIII) expression is elevated in the ventral ectoderm, a site of active BMP signal transduction. Moreover, overexpression of XeIF-4AIII induces epidermis in dissociated cells that would otherwise adopt a neural fate, mimicking the effects of BMP-4. Epidermal induction by XeIF-4AIII requires both an active BMP signaling pathway and an extracellular intermediate. Our results suggest that XeIF-4AIII can regulate changes in cell fate through selective mRNA translation. We propose that BMPs and XeIF-4AIII interact through a positive feedback loop in the ventral ectoderm of the vertebrate gastrula.

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, overexpression 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 ectodermal 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.

Despite extensive study following the pioneering work of Spemann on lens development (Spemann, H. (1901) Verh. Anat. Ges. 15, 61-79) and the subsequent establishment of the concept of embryonic induction, the molecular mechanism of vertebrate lens induction remains largely unknown. Here we report that in Xenopus expression of Pax-6 results in lens formation in a cell autonomous manner. In animal cap experiments, Pax-6 induced expression of the lens-specific marker beta B1-crystallin without inducing the general neural marker NCAM. Ectopic Pax-6 expression also resulted in the formation of ectopic lenses in whole embryos as well as in animal cap explants indicating that in vertebrates, as well as Drosophila (Halder, G., Callaerts, P., and Gehring, W.J. (1995) Science 267, 1788-1792), Pax-6 can direct the development of major components of the eye. Interestingly, ectopic lenses formed in whole embryos without association with neural tissue. Treatments giving rise to anterior neural tissue in animal cap explants resulted in the expression of both beta B1-crystallin and Pax-6. Given the ability of Pax-6 to direct lens formation, we propose that the establishment of Pax-6 expression in the presumptive lens ectoderm during normal development is likely to be a critical response of lens-competent ectoderm to early lens inducers.

The Smad proteins have been implicated in the intracellular signaling of transforming growth factor-beta (TGF-beta) 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.

At gastrulation, vertebrate ectoderm is competent to differentiate into either neural tissue or epidermis. Several soluble factors that can neuralize ectoderm in explant cultures have been isolated. Alternatively, neuralization can be achieved by dissociating the cells of the blastula ectoderm. These various treatments appear to neuralize by blocking or diluting out the action of an epidermal-inducing factor. Recent results demonstrate that bone morphogenetic protein 4 (BMP-4), a member of the transforming growth factor beta (TGF-beta) ligand superfamily, is a potent neural inhibitor and epidermal inducer and may represent the endogenous epidermal-inducing factor.

Activins and other ligands in the TGFbeta 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.

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).

Cellular communication in the developing embryo is mediated by receptor-ligand interactions at the cell surface. Receptor protein tyrosine kinases (RTKs) have been shown to play a critical role in the development of the vertebrate embryo. The eph receptors are a large subclass of RTKs for which a corresponding ligand family has only recently been described. The restricted expression patterns of several eph receptors imply roles for these molecules in early vertebrate development. We have isolated both a ligand of the eph ligand family (ELF), that we have named XELF-a, and an eph-related receptor, XE10, the likely homolog of the murine eck/Sek-2 receptor. At least two forms of the XELF-a transcript are present in the developing embryo. A truncated form of the XELF-a ligand, XELF-á, is the first ELF ligand isolated that lacks both the membrane-spanning and membrane-anchoring motifs conserved among this family, suggesting that ELF ligands can function as fully soluble molecules in vivo. XELF-a and XE10 are expressed maternally and throughout early embryogenesis, while XELF-á is only expressed zygotically. The dynamic expression patterns of these signalling molecules, in both mesoderm and neurectoderm, suggest that they may play a role in the patterning of the early vertebrate embryo.

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 neuralized 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 prospective anterior neural fate in the vertebrate embryo.