PV.1 Suppresses the Expression of FoxD5b during Neural Induction in Xenopus Embryos

Suppression of bone morphogenetic protein (BMP) signaling induces neural induction in the ectoderm of developing embryos. BMP signaling inhibits eural induction via the expression of various neural suppressors. Previous research has demonstrated that the ectopic expression of dominant negative BMP receptors (DNBR) reduces the expression of target genes down-stream of BMP and leads to neural induction. Additionally, gain-of-function experiments have shown that BMP downstream target genes such as MSX1, GATA1b and Vent are involved in the suppression of neural induction. For example, the Vent1/2 genes are involved in the suppression of Geminin and Sox3 expression in the neural ectodermal region of embryos. In this paper, we investigated whether PV.1, a BMP downstream target gene, negatively regulates the expression of FoxD5b, which plays a role in maintaining a neural progenitor population. A promoter assay and a cyclohexamide experiment demonstrated that PV.1 negatively regulates FoxD5b expression.     

Neural induction requires BMP inhibition only as a late step, and involves signals other than FGF and Wnt antagonists

A dominant molecular explanation for neural induction is the 'default model', which proposes that the ectoderm is pre-programmed towards a neural fate, but is normally inhibited by endogenous BMPs. Although there is strong evidence favouring this in Xenopus, data from other organisms suggest more complexity, including an involvement of FGF and modulation of Wnt. However, it is generally believed that these additional signals also act by inhibiting BMPs. We have investigated whether BMP inhibition is necessary and/or sufficient for neural induction. In the chick, misexpression of BMP4 in the prospective neural plate inhibits the expression of definitive neural markers (Sox2 and late Sox3), but does not affect the early expression of Sox3, suggesting that BMP inhibition is required only as a late step during neural induction. Inhibition of BMP signalling by the potent antagonist Smad6, either alone or together with a dominant-negative BMP receptor, Chordin and/or Noggin in competent epiblast is not sufficient to induce expression of Sox2 directly, even in combination with FGF2, FGF3, FGF4 or FGF8 and/or antagonists of Wnt signalling. These results strongly suggest that BMP inhibition is not sufficient for neural induction in the chick embryo. To test this in Xenopus, Smad6 mRNA was injected into the A4 blastomere (which reliably contributes to epidermis but not to neural plate or its border) at the 32-cell stage: expression of neural markers (Sox3 and NCAM) is not induced. We propose that neural induction involves additional signalling events that remain to be identified.     

Cell fate specification and competence by Coco,a maternal BMP,TGFbeta and Wnt inhibitor

Patterning of the pre-gastrula embryo and subsequent neural induction post-gastrulation are very complex and intricate processes of which little, until recently, has been understood. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMP signaling is sufficient for neural induction. Many secreted BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo and therefore are predicted to act as bona fide endogenous neural inducers. Other cell-autonomous inhibitors of the BMP pathway are more widely expressed, such as the inhibitory Smads, Smad6 and Smad7. In this report we describe the biological and biochemical characterization of 51-B6, a novel member of Cerberus/Dan family of secreted BMP inhibitors, which we identified in a screen for Smad7-induced genes. This gene is expressed maternally in an animal to vegetal gradient, and its expression levels decline rapidly following gastrulation. In contrast to known BMP inhibitors, 51-B6 is broadly expressed in the ectoderm until the end of gastrulation. The timing, pattern of expression, and activities of this gene makes it unique when compared to other BMP/TGFbeta/Wnt secreted inhibitors which are expressed only zygotically and maintained post-gastrulation. We propose that a function of 51-B6 is to block BMP and TGFbeta signals in the ectoderm in order to regulate cell fate specification and competence prior to the onset of neural induction. In addition, we demonstrate that 51-B6 can act as a neural inducer and induce ectopic head-like structures in neurula staged embryos. Because of this embryological activity, we have renamed this clone Coco, after the Spanish word meaning head.     

Endogenous patterns of BMP signaling during early chick development

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily signaling molecules that play important roles in a wide variety of developmental processes. In this study, we have used an antibody specific for the phosphorylated and activated form of Smad1 to examine endogenous patterns of BMP signaling in chick embryos during early development. We find complex spatial and temporal distributions of BMP signaling that elucidate how BMPs may function in multiple patterning events in the early chick embryo. In the pregastrula embryo, we find that BMP signaling is initially ubiquitous and is extinguished in the epiblast at the onset of primitive streak formation. At the head process stage, BMP signaling is inactivated in prospective neural plate, while it is strongly activated at the neural plate border, a region which is populated by cells that will give rise to neural crest. During later development, we find a dynamic spatiotemporal activation of BMP signaling along the rostrocaudal axis, in the dorsal neural tube, in the notochord, and in the somites during their maturation process. We discuss the implication of our results for endogenous functions of BMP signaling during chick development.     

Bone morphogenetic protein acts as a ventral mesoderm modifier in early Xenopus embryos

Mesoderm of early vertebrate embryos gradually acquires dorsal–ventral polarity during embryogenesis. This specification of mesoderm is thought to be regulated by several polypeptide growth factors. Bone morphogenetic protein (BMP), a member of the TGF-β family, is one of the regulators suggested to be involved in the formation of ventral mesoderm. In this paper, the nature of the endogenous BMP signal in dorsal–ventral specification was assessed in early Xenopus embryos using a dominant negative mutant of the Xenopus BMP receptor. In ectodermal explant assays, disruption of endogenous BMP signaling by the mutant receptor changed the competence of the explant cells to mesoderm-inducing factors, activin and basic fibroblast growth factor (bFGF), and led to formation of neural tissue without mesoderm induction. This result suggests that endogenous BMP acts as a ventral mesoderm modifier rather than a ventral mesoderm inducer, and that interactions between endogenous BMP and mesoderm-inducing factors may be important in dorsal–ventral patterning of embryonic mesoderm. In addition, the induction of neural tissue by inhibition of the BMP signaling pathway also suggests involvement of BMP in neural induction.     

A gene with sequence similarity to Drosophila engrailed is expressed during the development of the neural tube and vertebrae in the mouse

The mouse genes En-1 and En-2 display sequence similarity, in and around the homeobox region, to the engrailed family in Drosophila. This paper describes their pattern of expression in the 12.5-day mouse embryo as determined by in situ hybridization. En-2 is expressed in a subset of cells expressing En-1. Both genes are expressed in the developing midbrain and its junction with the hindbrain. In addition, En-1 is expressed in the floor of the hindbrain, a restricted ventrolateral segment of the neural tube throughout the trunk and anterior part of the tail, the dermatome of tail somites, the centrum and costal processes in developing vertebrae, a restricted region of facial mesenchyme and the limb-bud ectoderm. Supplementary studies of 9.5-day and 10.5-day embryos showed that the same pattern of expression pertained in the neural tube, but that expression in the somites is at first confined to the dermatome and later found at a low level in restricted sclerotomal regions. Both genes are expressed in restricted domains which do not cross tissue-type boundaries. In several instances, however, boundaries of expression lie within morphologically undifferentiated tissue. These results suggest that En-1 and En-2 may be involved in the establishment or maintenance of the spatial integrity of specific domains within developing tissues.     

BMP antagonists and FGF signaling contribute to different domains of the neural plate in Xenopus

In ectodermal explants from Xenopus embryos, inhibition of BMP signaling is sufficient for neural induction, leading to the idea that neural fate is the default state in the ectoderm. Many of these experiments assayed the action of BMP antagonists on animal caps, which are relatively naïve explants of prospective ectoderm, and different results have led to debate regarding both the mechanism of neural induction and the appropriateness of animal caps as an assay system. Here we address whether BMP antagonists are only able to induce neural fates in pre-patterned explants, and the extent to which neural induction requires FGF signaling. We suggest that some discrepancies in conclusion depend on the interpretations of sox gene expression, which we show not only marks definitive neural tissue, but also tissue that is not yet committed to neural fates. Part of the early sox2 domain requires FGF signaling, but in the absence of organizer signaling, this domain reverts to epidermal fates. We also reinforce the evidence that ectodermal explants are naïve, and that explants that lack any dorsal prepattern are readily neuralized by BMP antagonists, even when FGF signaling is inhibited.     

Relationship between Wnt-1 and En-2 expression domains during early development of normal and ectopic met-mesencephalon.

Grafting a met-mesencephalic portion of neural tube from a 9.5-day mouse embryo into the prosencephalon of a 2-day chick embryo results in the induction of chick En-2 (ChickEn) expression in cells in contact with the graft (Martinez et al., 1991). In this paper we investigate the possibility of Wnt-1 being one of the factors involved in En-2 induction. Since Wnt-1 and En-2 expression patterns have been described as diverging during development of the met-mesencephalic region, we first compared Wnt-1 and En-2 expression in this domain by in situ hybridization in mouse embryos after embryonic day 8.5. A ring of Wnt-1-expressing cells is detected encircling the neural tube in the met-mesencephalic region at least until day 12.5. This ring consistently overlapped with the En-2 expression domain, and corresponds to the position of this latter gene's maximal expression. We subsequently studied ChickEn ectopic induction in chick embryos grafted with various portions of met-mesencephalon. When the graft originated from the level of the Wnt-1-positive ring, ChickEn induction was observed in 71% of embryos, and in these cases correlated with Wnt-1 expression in the grafted tissue. In contrast, this percentage dropped significantly when the graft was taken from more rostral or caudal parts of the mesencephalic vesicle. Taken together, these results are compatible with a prolonged role of Wnt-1 in the specification and/or development of the met-mesencephalic region, and show that Wnt-1 could be directly or indirectly involved in the regulation of En-2 expression around the Wnt-1-positive ring during this time. We also provide data on the position of the Wnt-1-positive ring relative to anatomical boundaries in the neural tube, which suggest a more general role for the Wnt-1 protein as a positional signal involved in organizing the met-mesencephalic domain.     

Threshold-dependent BMP-mediated repression: a model for a conserved mechanism that patterns the neuroectoderm

Subdivision of the neuroectoderm into three rows of cells along the dorsal-ventral axis by neural identity genes is a highly conserved developmental process. While neural identity genes are expressed in remarkably similar patterns in vertebrates and invertebrates, previous work suggests that these patterns may be regulated by distinct upstream genetic pathways. Here we ask whether a potential conserved source of positional information provided by the BMP signaling contributes to patterning the neuroectoderm. We have addressed this question in two ways: First, we asked whether BMPs can act as bona fide morphogens to pattern the Drosophila neuroectoderm in a dose-dependent fashion, and second, we examined whether BMPs might act in a similar fashion in patterning the vertebrate neuroectoderm. In this study, we show that graded BMP signaling participates in organizing the neural axis in Drosophila by repressing expression of neural identity genes in a threshold-dependent fashion. We also provide evidence for a similar organizing activity of BMP signaling in chick neural plate explants, which may operate by the same double negative mechanism that acts earlier during neural induction. We propose that BMPs played an ancestral role in patterning the metazoan neuroectoderm by threshold-dependent repression of neural identity genes.     

Neural induction in Xenopus requires early FGF signalling in addition to BMP inhibition

Neural induction constitutes the first step in the generation of the vertebrate nervous system from embryonic ectoderm. Work with Xenopus ectodermal explants has suggested that epidermis is induced by BMP signals, whereas neural fates arise by default following BMP inhibition. In amniotes and ascidians, however, BMP inhibition does not appear to be sufficient for neural fate acquisition, which is initiated by FGF signalling. We decided to re-evaluate in the context of the whole embryo the roles of the BMP and FGF pathways during neural induction in Xenopus. We find that ectopic BMP activity converts the neural plate into epidermis, confirming that this pathway must be inhibited during neural induction in vivo. Conversely, inhibition of BMP, or of its intracellular effector SMAD1 in the non-neural ectoderm leads to epidermis suppression. In no instances, however, is BMP/SMAD1 inhibition sufficient to elicit neural induction in ventral ectoderm. By contrast, we find that neural specification occurs when weak eFGF or low ras signalling are combined with BMP inhibition. Using all available antimorphic FGF receptors (FGFR), as well as the pharmacological FGFR inhibitor SU5402, we demonstrate that pre-gastrula FGF signalling is required in the ectoderm for the emergence of neural fates. Finally, we show that although the FGF pathway contributes to BMP inhibition, as in other model systems, it is also essential for neural induction in vivo and in animal caps in a manner that cannot be accounted for by simple BMP inhibition. Taken together, our results reveal that in contrast to predictions from the default model, BMP inhibition is required but not sufficient for neural induction in vivo. This work contributes to the emergence of a model whereby FGF functions as a conserved initiator of neural specification among chordates.     

A role for iro1 and iro7 in the establishment of an anteroposterior compartment of the ectoderm adjacent to the midbrain-hindbrain boundary

We have identified a novel Iroquois (Iro) gene, iro7, in zebrafish. iro7 is expressed during gastrulation along with iro1 in a compartment of the dorsal ectoderm that includes the prospective midbrain-hindbrain domain, the adjacent neural crest and the trigeminal placodes in the epidermis. The iro1 and iro7 expression domain is expanded in headless and masterblind mutants, which are characterized by exaggerated Wnt signaling. Early expansion of iro1 and iro7 expression in these mutants correlates with expansion of the midbrain-hindbrain boundary (MHB) domain, the neural crest and trigeminal neurons, raising the possibility that iro1 and iro7 have a role in determination of these ectodermal derivatives. A knockdown of iro7 function revealed that iro7 is essential for the determination of neurons in the trigeminal placode. In addition, a knockdown of both iro1 and iro7 genes uncovered their essential roles in neural crest development and establishment of the isthmic organizer at the MHB. These results suggest a new role for Iro genes in establishment of an ectodermal compartment after Wnt signaling in vertebrate development. Furthermore, analysis of activator or repressor forms of iro7 suggests that iro1 and iro7 are likely to function as repressors in establishment of the isthmic organizer and neural crest, and Iro genes may have dual functions as repressors and activators in neurogenesis.     

Early neural crest induction requires an initial inhibition of Wnt signals

Neural crest (NC) induction is a long process that continues through gastrula and neurula stages. In order to reveal additional stages of NC induction we performed a series of explants where different known inducing tissues were taken along with the prospective NC. Interestingly the dorso-lateral marginal zone (DLMZ) is only able to promote the expression of a subset of neural plate border (NPB) makers without the presence of specific NC markers. We then analysed the temporal requirement for BMP and Wnt signals for the NPB genes Hairy2a and Dlx5, compared to the expression of neural plate (NP) and NC genes. Although the NP is sensitive to BMP levels at early gastrula stages, Hairy2a/Dlx5 expression is unaffected. Later, the NP becomes insensitive to BMP levels at late gastrulation when NC markers require an inhibition. The NP requires an inhibition of Wnt signals prior to gastrulation, but becomes insensitive during early gastrula stages when Hairy2a/Dlx5 requires an inhibition of Wnt signalling. An increase in Wnt signalling is then important for the switch from NPB to NC at late gastrula stages. In addition to revealing an additional distinct signalling event in NC induction, this work emphasizes the importance of integrating both timing and levels of signalling activity during the patterning of complex tissues such as the vertebrate ectoderm.     

Neural induction. A bird's eye view

Since the discovery of the phenomenon of neural induction by Spemann and Mangold in 1924, considerable effort has been invested in identifying the signals produced by the organizer that are responsible for diverting the fate of cells from epidermal to neural. Substantial progress has been made only recently by the finding in amphibians that BMP4 is a neural inhibitor and epidermal inducer, and that endogenous antagonists of BMPs are secreted by the organizer. However, recent results in the chick point to the existence of other, upstream events required before BMP inhibition stabilizes neural fates. Here we take a critical view of the evidence for and against the view that BMP inhibition is a sufficient trigger for neural induction in different vertebrates.     

Neural induction requires continued suppression of both Smad1 and Smad2 signals during gastrulation

Vertebrate neural induction requires inhibition of bone morphogenetic protein (BMP) signaling in the ectoderm. However, whether inhibition of BMP signaling is sufficient to induce neural tissues in vivo remains controversial. Here we have addressed why inhibition of BMP/Smad1 signaling does not induce neural markers efficiently in Xenopus ventral ectoderm, and show that suppression of both Smad1 and Smad2 signals is sufficient to induce neural markers. Manipulations that inhibit both Smad1 and Smad2 pathways, including a truncated type IIB activin receptor, Smad7 and Ski, induce early neural markers and inhibit epidermal genes in ventral ectoderm; and co-expression of BMP inhibitors with a truncated activin/nodal-specific type IB activin receptor leads to efficient neural induction. Conversely, stimulation of Smad2 signaling in the neural plate at gastrula stages results in inhibition of neural markers, disruption of the neural tube and reduction of head structures, with conversion of neural to neural crest and mesodermal fates. The ability of activated Smad2 to block neural induction declines by the end of gastrulation. Our results indicate that prospective neural cells are poised to respond to Smad2 and Smad1 signals to adopt mesodermal and non-neural ectodermal fates even at gastrula stages, after the conventionally assigned end of mesodermal competence, so that continued suppression of both mesoderm- and epidermis-inducing Smad signals leads to efficient neural induction.