Notch1 is asymmetrically distributed from the beginning of embryogenesis and controls the ventral center.

Based on functional evidence, we have previously demonstrated that early ventral Notch1 activity restricts dorsoanterior development in Xenopus We found that Notch1 has ventralizing properties and abolishes the dorsalizing activity of ß-catenin by reducing its steady state levels, in a process that does not require ß-catenin phosphorylation by glycogen synthase kinase 3ß. In the present work, we demonstrate that Notch1 mRNA and protein are enriched in the ventral region from the beginning of embryogenesis in Xenopus This is the earliest sign of ventral development, preceding the localized expression of wnt8a, bmp4 and Ventx genes in the ventral center and the dorsal accumulation of nuclear ß-catenin. Knockdown experiments indicate that Notch1 is necessary for the normal expression of genes essential for ventral-posterior development. These results indicate that during early embryogenesis ventrally located Notch1 promotes the development of the ventral center. Together with our previous evidence, these results suggest that ventral enrichment of Notch1 underlies the process by which Notch1 participates in restricting nuclear accumulation of ß-catenin to the dorsal side.

Notch destabilises maternal beta-catenin and restricts dorsal-anterior development in Xenopus.

The blastula chordin- and noggin-expressing centre (BCNE) is the predecessor of the Spemann-Mangold's organiser and also contains the precursors of the brain. This signalling centre comprises animal-dorsal and marginal-dorsal cells and appears as a consequence of the nuclear accumulation of ß-catenin on the dorsal side. Here, we propose a role for Notch that was not previously explored during early development in vertebrates. Notch initially destabilises ß-catenin in a process that does not depend on its phosphorylation by GSK3. This is important to restrict the BCNE to its normal extent and to control the size of the brain.

A maternal dorsoventral prepattern revealed by an asymmetric distribution of ventralizing molecules before fertilization in Xenopus laevis.

The establishment of the embryonic dorsoventral axis in Xenopus occurs when the radial symmetry around the egg's animal-vegetal axis is broken to give rise to the typical symmetry of Bilaterians. We have previously shown that the Notch1 protein is ventrally enriched during early embryogenesis in Xenopus laevis and zebrafish and exerts ventralizing activity through ß-Catenin destabilization and the positive regulation of ventral center genes in X. laevis. These findings led us to further investigate when these asymmetries arise. In this work, we show that the asymmetrical distribution of Notch1 protein and mRNA precedes cortical rotation and even fertilization in X. laevis. Moreover, we found that in unfertilized eggs transcripts encoded by the ventralizing gene bmp4 are also asymmetrically distributed in the animal hemisphere and notch1 transcripts accumulate consistently on the same side of the eccentric maturation point. Strikingly, a Notch1 asymmetry orthogonal to the animal-vegetal axis appears during X. laevis oogenesis. Thus, we show for the first time a maternal bias in the distribution of molecules that are later involved in ventral patterning during embryonic axialization, strongly supporting the hypothesis of a dorsoventral prepattern or intrinsic bilaterality of Xenopus eggs before fertilization.

Nodal and <i>churchill1</i> position the expression of a notch ligand during <i>Xenopus</i> germ layer segregation.

In vertebrates, Nodal signaling plays a major role in endomesoderm induction, but germ layer delimitation is poorly understood. In avian embryos, the neural/mesoderm boundary is controlled by the transcription factor CHURCHILL1, presumably through the repressor ZEB2, but there is scarce knowledge about its role in other vertebrates. During amphibian gastrulation, Delta/Notch signaling refines germ layer boundaries in the marginal zone, but it is unknown the place this pathway occupies in the network comprising Churchill1 and Nodal. Here, we show that <i>Xenopus churchill1</i> is expressed in the presumptive neuroectoderm at mid-blastula transition and during gastrulation, upregulates <i>zeb2</i>, prevents <i>dll1</i> expression in the neuroectoderm, and favors neuroectoderm over endomesoderm development. Nodal signaling prevents <i>dll1</i> expression in the endoderm but induces it in the presumptive mesoderm, from where it activates Notch1 and its target gene <i>hes4</i> in the non-involuting marginal zone. We propose a model where Nodal and Churchill1 position Dll1/Notch1/Hes4 domains in the marginal zone, ensuring the delimitation between mesoderm and neuroectoderm.

Delta-Notch signaling is involved in the segregation of the three germ layers in Xenopus laevis.

In vertebrates, the induction of the three germ layers (ectoderm, mesoderm and endoderm) has been extensively studied, but less is known about how they segregate. Here, we investigated whether Delta-Notch signaling is involved in this process. Activating the pathway in the marginal zone with Notch(ICD) resulted in an expansion of endodermal and neural ectoderm precursors, leaving a thinner mesodermal ring around the blastopore at gastrula stage, when germ layers are segregated. On the other hand, when the pathway was blocked with Delta-1(STU) or with an antisense morpholino oligonucleotide against Notch, the pan-mesodermal brachyury (bra) domain was expanded and the neural border was moved animalwards. Strikingly, the suprablastoporal endoderm was either expanded when Delta-1 signaling was blocked, or reduced after the general knock-down of Notch. In addition, either activating or blocking the pathway delays the blastopore closure. We conclude that the process of delimiting the three germ layers requires Notch signaling, which may be finely regulated by ligands and/or involve non-canonical components of the pathway. Moreover, Notch activity must be modulated at appropriate levels during this process in order to keep normal morphogenetic movements during gastrulation.

Segregation of brain and organizer precursors is differentially regulated by Nodal signaling at blastula stage.

The blastula Chordin- and Noggin-expressing (BCNE) center comprises animal-dorsal and marginal-dorsal cells of the amphibian blastula and contains the precursors of the brain and the gastrula organizer. Previous findings suggested that the BCNE behaves as a homogeneous cell population that only depends on nuclear ß-catenin activity but does not require Nodal and later segregates into its descendants during gastrulation. In contrast to previous findings, in this work, we show that the BCNE does not behave as a homogeneous cell population in response to Nodal antagonists. In fact, we found that chordin.1 expression in a marginal subpopulation of notochordal precursors indeed requires Nodal input. We also establish that an animal BCNE subpopulation of cells that express both, chordin.1 and sox2 (a marker of pluripotent neuroectodermal cells), and gives rise to most of the brain, persisted at blastula stage after blocking Nodal. Therefore, Nodal signaling is required to define a population of chordin.1+ cells and to restrict the recruitment of brain precursors within the BCNE as early as at blastula stage. We discuss our findings in Xenopus in comparison to other vertebrate models, uncovering similitudes in early brain induction and delimitation through Nodal signaling.

Glyphosate-based herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling.

The broad spectrum herbicide glyphosate is widely used in agriculture worldwide. There has been ongoing controversy regarding the possible adverse effects of glyphosate on the environment and on human health. Reports of neural defects and craniofacial malformations from regions where glyphosate-based herbicides (GBH) are used led us to undertake an embryological approach to explore the effects of low doses of glyphosate in development. Xenopus laevis embryos were incubated with 1/5000 dilutions of a commercial GBH. The treated embryos were highly abnormal with marked alterations in cephalic and neural crest development and shortening of the anterior-posterior (A-P) axis. Alterations on neural crest markers were later correlated with deformities in the cranial cartilages at tadpole stages. Embryos injected with pure glyphosate showed very similar phenotypes. Moreover, GBH produced similar effects in chicken embryos, showing a gradual loss of rhombomere domains, reduction of the optic vesicles, and microcephaly. This suggests that glyphosate itself was responsible for the phenotypes observed, rather than a surfactant or other component of the commercial formulation. A reporter gene assay revealed that GBH treatment increased endogenous retinoic acid (RA) activity in Xenopus embryos and cotreatment with a RA antagonist rescued the teratogenic effects of the GBH. Therefore, we conclude that the phenotypes produced by GBH are mainly a consequence of the increase of endogenous retinoid activity. This is consistent with the decrease of Sonic hedgehog (Shh) signaling from the embryonic dorsal midline, with the inhibition of otx2 expression and with the disruption of cephalic neural crest development. The direct effect of glyphosate on early mechanisms of morphogenesis in vertebrate embryos opens concerns about the clinical findings from human offspring in populations exposed to GBH in agricultural fields.

Notch signaling in the division of germ layers in bilaterian embryos.

Bilaterian embryos are triploblastic organisms which develop three complete germ layers (ectoderm, mesoderm, and endoderm). While the ectoderm develops mainly from the animal hemisphere, there is diversity in the location from where the endoderm and the mesoderm arise in relation to the animal-vegetal axis, ranging from endoderm being specified between the ectoderm and mesoderm in echinoderms, and the mesoderm being specified between the ectoderm and the endoderm in vertebrates. A common feature is that part of the mesoderm segregates from an ancient bipotential endomesodermal domain. The process of segregation is noisy during the initial steps but it is gradually refined. In this review, we discuss the role of the Notch pathway in the establishment and refinement of boundaries between germ layers in bilaterians, with special focus on its interaction with the Wnt/ß-catenin pathway.

FoxA4 favours notochord formation by inhibiting contiguous mesodermal fates and restricts anterior neural development in Xenopus embryos.

In vertebrates, the embryonic dorsal midline is a crucial signalling centre that patterns the surrounding tissues during development. Members of the FoxA subfamily of transcription factors are expressed in the structures that compose this centre. Foxa2 is essential for dorsal midline development in mammals, since knock-out mouse embryos lack a definitive node, notochord and floor plate. The related gene foxA4 is only present in amphibians. Expression begins in the blastula -chordin and -noggin expressing centre (BCNE) and is later restricted to the dorsal midline derivatives of the Spemann's organiser. It was suggested that the early functions of mammalian foxa2 are carried out by foxA4 in frogs, but functional experiments were needed to test this hypothesis. Here, we show that some important dorsal midline functions of mammalian foxa2 are exerted by foxA4 in Xenopus. We provide new evidence that the latter prevents the respecification of dorsal midline precursors towards contiguous fates, inhibiting prechordal and paraxial mesoderm development in favour of the notochord. In addition, we show that foxA4 is required for the correct regionalisation and maintenance of the central nervous system. FoxA4 participates in constraining the prospective rostral forebrain territory during neural specification and is necessary for the correct segregation of the most anterior ectodermal derivatives, such as the cement gland and the pituitary anlagen. Moreover, the early expression of foxA4 in the BCNE (which contains precursors of the whole forebrain and most of the midbrain and hindbrain) is directly required to restrict anterior neural development.

An intact brachyury function is necessary to prevent spurious axial development in Xenopus laevis.

We have previously shown that the member of the HES family hairy2 induces the ectopic expression of dorsal markers when it is overexpressed in the ventral side of Xenopus embryos. Intriguingly, hairy2 represses the mesoderm transcription factor brachyury (bra) throughout its domain in the marginal zone. Here we show that in early gastrula, bra and hairy2 are expressed in complementary domains. Overexpression of bra repressed hairy2. Interference of bra function with a dominant-negative construct expanded the hairy2 domain and, like hairy2 overexpression, promoted ectopic expression of dorsal axial markers in the ventral side and induced secondary axes without head and notochord. Hairy2 depletion rescued the ectopic dorsal development induced by interference of bra function. We concluded that an intact bra function is necessary to exclude hairy2 expression from the non-organiser field, to impede the ectopic specification of dorsal axial fates and the appearance of incomplete secondary axes. This evidence supports a previously unrecognised role for bra in maintaining the dorsal fates inhibited in the ventral marginal zone, preventing the appearance of trunk duplications.

The Notch-target gene hairy2a impedes the involution of notochordal cells by promoting floor plate fates in Xenopus embryos.

We have previously shown that the early Xenopus organiser contains cells equally potent to give rise to notochord or floor plate, and that Notch signalling triggers a binary decision, favouring the floor plate fate at the expense of the notochord. Now, we present evidence that Delta1 is the ligand that triggers the binary switch, which is executed through the Notch-mediated activation of hairy2a in the surrounding cells within the organiser, impeding their involution through the blastopore and promoting their incorporation into the hairy2a+ notoplate precursors (future floor-plate cells) in the dorsal non-involuting marginal zone.

Notch activates sonic hedgehog and both are involved in the specification of dorsal midline cell-fates in Xenopus.

We analysed the role of Notch signalling during the specification of the dorsal midline in Xenopus embryos. By activating or blocking the pathway we found that Notch expands the floor plate domain of sonic hedgehog and pintallavis and represses the notochordal markers chordin and brachyury, with a concomitant reduction of the notochord size. We propose that within a population of the early organiser with equivalent potential to develop either as notochord or floor plate, Notch activation favours floor plate development at the expense of the notochord, preferentially before mid gastrula. We present evidence that sonic hedgehog down-regulates chordin, suggesting that secreted Sonic hedgehog may be involved or reinforcing the cell-fate switch executed by Notch. We also show that Notch signalling requires Presenilin to modulate this switch.