Left-right patterning of the mouse lateral plate requires nodal produced in the node

Initial determination of left-right (L-R) polarity in mammalian embryos takes place in the node. However, it is not known how asymmetric signals are generated in the node and transferred to the lateral plate mesoderm (LPM). Mice homozygous for a hypomorphic Nodal allele (Nodal(neo)) were generated and found to exhibit L-R defects, including right isomerism. Although the mutant embryos express Nodal at gastrulation stages, the subsequent expression of this gene in the node and left LPM is lost. A transgene that conferred Nodal expression specifically in the node rescued the L-R defects of the Nodal(neo/neo) embryos. Conversely, ectopic expression of the Nodal inhibitor Lefty2 in the node of Nodal(neo/+) embryos resulted in a phenotype similar to that of the Nodal(neo/neo) mutant. These results indicate that Nodal produced in the node is required for expression of Nodal and other left side-specific genes in the LPM.     

A role for the hypoblast (AVE) in the initiation of neural induction, independent of its ability to position the primitive streak

The mouse anterior visceral endoderm (AVE) has been implicated in embryonic polarity: it helps to position the primitive streak and some have suggested that it might act as a "head organizer", inducing forebrain directly. Here we explore the role of the hypoblast (the chick equivalent of the AVE) in the early steps of neural induction and patterning. We report that the hypoblast can induce a set of very early markers that are later expressed in the nervous system and in the forebrain, but only transiently. Different combinations of signals are responsible for different aspects of this early transient induction: FGF initiates expression of Sox3 and ERNI, retinoic acid can induce Cyp26A1 and only a combination of low levels of FGF8 together with Wnt- and BMP-antagonists can induce Otx2. BMP- and Wnt-antagonists and retinoic acid, in different combinations, can maintain the otherwise transient induction of these markers. However, neither the hypoblast nor any of these factors or combinations thereof can induce the definitive neural marker Sox2 or the formation of a mature neural plate or a forebrain, suggesting that the hypoblast is not a head organizer and that other signals remain to be identified. Interestingly, FGF and retinoids, generally considered as caudalizing factors, are shown here to play a role in the induction of a transient "pre-neural/pre-forebrain" state.     

The FERM protein Epb4.1l5 is required for organization of the neural plate and for the epithelial-mesenchymal transition at the primitive streak of the mouse embryo

During early mouse development, a single-layered epithelium is transformed into the three germ layers that are the basis of the embryonic body plan. Here we describe an ENU-induced mutation, limulus (lulu), which disrupts gastrulation and the organization of all three embryonic germ layers. Positional cloning and analysis of additional alleles show that lulu is a null allele of the FERM-domain gene erythrocyte protein band 4.1-like 5 (Epb4.1l5). During gastrulation, some cells in lulu mutants are trapped in the primitive streak at an intermediate stage of the epithelial-mesenchymal transition; as a result, the embryos have very little paraxial mesoderm. Epithelial layers of the later lulu embryo are also disrupted: definitive endoderm is specified but does not form a gut tube, and the neural plate is broad and forms ectopic folds rather than closing to make the neural tube. In contrast to zebrafish and Drosophila, in which orthologs of Epb4.1l5 control the apical localization and activity of Crumbs proteins, mouse Crumbs proteins are localized normally to the apical surface of the lulu mutant epiblast and neural plate. However, the defects in both the lulu primitive streak and neural plate are associated with disruption of the normal organization of the actin cytoskeleton. We propose that mouse Lulu (Epb4.1l5) helps anchor the actin-myosin contractile machinery to the membrane to allow the dynamic rearrangements of epithelia that mediate embryonic morphogenesis.     

Loss of late primitive streak mesoderm and interruption of left-right morphogenesis in the Ednrb(s-1Acrg) mutant mouse

This study characterizes defects associated with abnormal mesoderm development in mouse embryos homozygous for the induced Ednrb(s-1Acrg) allele of the piebald deletion complex. The Ednrb(s-1Acrg) deletion results in recessive embryonic lethality and mutant embryos exhibit a truncated posterior body axis. The primitive streak and node become disfigured, consistent with evidence that cell migration is impaired in newly formed mesoderm. Additional defects related to mesoderm development include notochord degeneration, somite malformations, and abnormal vascular development. Arrested heart looping morphogenesis and a randomized direction of embryonic turning indicate that left-right development is also perturbed. The expression of nodal and leftb, Tgf-beta-related genes involved in a left-determinant signaling pathway, is variably lost in the left lateral plate mesoderm. Mutational analysis has demonstrated that Fgf8 and Brachyury (T) are required for normal mesoderm and left-right development and the asymmetric expression of nodal and leftb. Fgf8 expression in nascent mesoderm exiting the primitive streak is dramatically reduced in mutant embryos, and diminished T expression accompanies the progressive loss of paraxial, lateral, and primitive streak mesoderm. In contrast, axial mesoderm persists and T and nodal appear to be appropriately expressed in their specific domains in the node and notochord. We propose that this mutation disrupts a morphogenetic pathway, likely involving FGF signaling, important for the development of streak-derived posterior mesoderm and lateral morphogenesis.     

Distinct requirements for extra-embryonic and embryonic bone morphogenetic protein 4 in the formation of the node and primitive streak and coordination of left-right asymmetry in the mouse

In the mouse and chick embryo, the node plays a central role in generating left-right (LR) positional information. Using several different strategies, we provide evidence in the mouse that bone morphogenetic protein 4 (Bmp4) is required independently in two different sites for node morphogenesis and for LR patterning. Bmp4 expression in the trophoblast-derived extra-embryonic ectoderm is essential for the normal formation of the node and primitive streak. However, tetraploid chimera analysis demonstrates that Bmp4 made in epiblast-derived tissues is required for robust LR patterning, even when normal node morphology is restored. In the absence of embryonic Bmp4, the expression of left-side determinants such as Nodal and Lefty2 is absent in the left lateral plate mesoderm (LPM). Noggin-mediated inhibition of Bmp activity in cultured wild-type embryos results in suppression of Nodal expression in the LPM. Thus, unlike previous models proposed in the chick embryo in which Bmp4 suppresses left-sided gene expression, our results suggest that Bmp acts as a positive facilitator of the left-sided molecular cascade and is required for Nodal induction and maintenance in the left LPM.     

Excess methionine suppresses the methylation cycle and inhibits neural tube closure in mouse embryos

Suppression of one-carbon metabolism or insufficient methionine intake are suggested to increase risk of neural tube defects (NTD). Here, exogenous methionine unexpectedly caused frequent NTD in cultured mouse embryos. NTD were associated with reduced cranial mesenchyme cell density, which may result from a preceding reduction in proliferation. The abundance ratio of S-adenosylmethionine to S-adenosylhomocysteine was also decreased in treated embryos, suggesting methylation reactions may be suppressed. Such an effect is potentially causative as NTD were also observed when DNA methylation was specifically inhibited. Thus, reduced cranial mesenchyme density and impairment of critical methylation reactions may contribute to development of methionine-induced NTD.     

Cell lineage analysis of the primitive and visceral endoderm of mouse embryos cultured in vitro

Cell lineages of the primitive endoderm and the visceral endoderm of mouse embryos were examined by culturing whole embryos in vitro. The primitive endoderm and visceral endoderm cells could be labelled by incubation of embryos in a medium containing horse radish peroxidase (HRP). HRP localization was chased throughout the culture period. The results show that the visceral endoderm derives from the primitive endoderm, and the visceral endoderm forms only the extra-embryonic endoderm (yolk sac endoderm) of the conceptus. The definitive endoderm which is probably derived from the head process, newly appears on the ventral surface of the embryo.     

Initiation of sodium channel clustering at the node of Ranvier in the mouse optic nerve

Ion fluxes in mammalian myelinated axons are restricted to the nodes of Ranvier, where, in particular, voltage-gated Na⁺ channels are clustered at a high density. The node of Ranvier is separated from the internode by two distinct domains of the axolemma, the paranode and the juxtaparanode. Each axonal domain is characterized by the presence of a specific protein complex. Although oligodendrocytes and/or myelin membranes are believed to play some instructive roles in the organization of axonal domains, the mechanisms leading to their localized distribution are not well understood. In this paper we focused on the involvement of myelin sheaths in this domain organization and examined the distribution of axonal components in the optic nerves of wild type, hypomyelinating jimpy mice and demyelinating PLP transgenic mice. The results showed that the clustering of Na⁺ channels does not require junction-like structures to be formed between the glial processes and axons, but requires mature oligodendrocytes to be present in close vicinity.     

Neural induction and patterning in the mouse in the absence of the node and its derivatives

The signals which induce vertebrate neural tissue and pattern it along the anterior-posterior (A-P) axis have been proposed to emanate from Spemann's organizer, which in mammals is a structure termed the node. However, mouse embryos mutant for HNF3 beta lack a morphological node and node derivatives yet undergo neural induction. Gene expression domains occur at their normal A-P axial positions along the mutant neural tubes in an apparently normal temporal manner, including the most anterior and posterior markers. This neural patterning occurs in the absence of expression of known organizer genes, including the neural inducers chordin and noggin. Other potential signaling centers in gastrulating mutant embryos appear to express their normal constellation of putative secreted factors, consistent with the possibility that neural-inducing and -patterning signals emanate from elsewhere or at an earlier time. Nevertheless, we find that the node and the anterior primitive streak, from which the node derives, are direct sources of neural-inducing signals, as judged by expression of the early midbrain marker Engrailed, in explant-recombination experiments. Similar experiments showed the neural-inducing activity in HNF3 beta mutants to be diffusely distributed. Our results indicate that the mammalian organizer is capable of neural induction and patterning of the neural plate, but that maintenance of an organizer-like signaling center is not necessary for either process.     

Specification of neural crest cell formation and migration in mouse embryos

Of all the model organisms used to study human development, rodents such as mice most accurately reflect human craniofacial development. Collective advances in mouse embryology and mouse genetics continue to shape our understanding of neural crest cell development and by extrapolation the etiology of human congenital head and facial birth defects. The aim of this review is to highlight the considerable progress being made in our understanding of cranial neural crest cell patterning in mouse embryos.     

Incipient forebrain boundaries traced by differential gene expression and fate mapping in the chick neural plate

We correlated available fate maps for the avian neural plate at stages HH4 and HH8 with the progress of local molecular specification, aiming to determine when the molecular specification maps of the primary longitudinal and transversal domains of the anterior forebrain agree with the fate mapped data. To this end, we examined selected gene expression patterns as they normally evolved in whole mounts and sections between HH4 and HH8 (or HH10/11 in some cases), performed novel fate-mapping experiments within the anterior forebrain at HH4 and examined the results at HH8, and correlated grafts with expression of selected gene markers. The data provided new details to the HH4 fate map, and disclosed some genes (e.g., Six3 and Ganf) whose expression domains initially are very extensive and subsequently retract rostralwards. Apart from anteroposterior dynamics, some genes soon became downregulated at the prospective forebrain floor plate, or allowed to identify an early roof plate domain (dorsoventral pattern). Peculiarities of the telencephalon (initial specification and differentiation of pallium versus subpallium) are contemplated. The basic anterior forebrain subdivisions seem to acquire correlated specification and fate mapping patterns around stage HH8.