Nodal signaling patterns the organizer

Spemann's organizer plays an essential role in patterning the vertebrate embryo. During gastrulation, organizer cells involute and form the prechordal plate anteriorly and the notochord more posteriorly. The fate mapping and gene expression analyses in zebrafish presented in this study reveal that this anteroposterior polarity is already initiated in the organizer before gastrulation. Prechordal plate progenitors reside close to the blastoderm margin and express the homeobox gene goosecoid, whereas notochord precursors are located further from the margin and express the homeobox gene floating head. The nodal-related genes cyclops and squint are expressed at the blastoderm margin and are required for prechordal plate and notochord formation. We show that differential activation of the Nodal signaling pathway is essential in establishing anteroposterior pattern in the organizer. First, overexpression of cyclops and squint at different doses leads to the induction of floating head at low doses and the induction of both goosecoid and floating head at higher doses. Second, decreasing Nodal signaling using different concentrations of the antagonist Antivin inhibits goosecoid expression at low doses and blocks expression of both goosecoid and floating head at higher doses. Third, attenuation of Nodal signaling in zygotic mutants for the EGF-CFC gene one-eyed pinhead, an essential cofactor for Nodal signaling, leads to the loss of goosecoid expression and expansion of floating head expression in the organizer. Concomitantly, cells normally fated to become prechordal plate are transformed into notochord progenitors. Finally, activation of Nodal signaling at different times suggests that prechordal plate specification requires sustained Nodal signaling, whereas transient signaling is sufficient for notochord development. Together, these results indicate that differential Nodal signaling patterns the organizer before gastrulation, with the highest level of activity required for anterior fates and lower activity essential for posterior fates.     

Xenopus crescent encoding a Frizzled-like domain is expressed in the Spemann organizer and pronephros

The Spemann organizer can be subdivided into head- and trunk-inducing tissues along the anteroposterior axis (Mangold, 1933. Naturwiisenschaften 43, 761-766; Spemann, 1931. Wilhelm Roux Arch. Entwicklungsmech. Org. 123, 389-517). Recent studies have suggested that head formation is brought about by repression of both Wnt and BMP signalling (Glinka et al., 1998. Nature 391, 357-362; Glinka et al., 1997. Nature 389, 517-519). Several Wnt inhibitors secreted from the head organizer region have been identified in Xenopus, such as Cerberus (Bouwmeester et al., 1996. Nature 382, 595-601), Frzb-1 (Leyns et al., 1997. Cell 88, 747-756; Lin et al., 1997. Proc. Natl. Acad. Sci. USA 94, 11196-11200), and Dkk-1 (Glinka et al., 1998. Nature 391, 357-362), supporting this two-inhibitor model. To isolate genes expressed in the head organizer, we screened a prechordal plate cDNA library by sequencing and expression pattern, and isolated the Xenopus ortholog of chick crescent encoding a Frizzled-like domain that is related to Wnt-binding regions of the Frizzled-family proteins. Expression of Xenopus crescent was first detected in the Spemann organizer region at the early gastrula stage and later in prechordal plate cells lining the boundary of mesoderm and ectoderm layers and in the anterior endoderm. At tailbud stages, the expression in the endomesoderm region was diminished, while expression in the pronephros became detectable. In animal cap assays, crescent gene was synergistically upregulated by coexpression of Xlim1, Ldb1, and Siamois, but not by Activin treatment.     

The EGF-CFC protein one-eyed pinhead is essential for nodal signaling

The zebrafish EGF-CFC gene one-eyed pinhead (oep) is required zygotically for the formation of the ventral neuroectoderm, endoderm, and prechordal plate. Here we report that embryos lacking both maternal and zygotic Oep activity are defective in germ layer formation, organizer development, and the positioning of the anterior-posterior axis. An identical phenotype is displayed by double mutants for the nodal-related genes squint and cyclops. Mutations in oep eliminate the response to Squint and Cyclops overexpression but are suppressed by expression of Activin and activated forms of the type I receptor ActRIB and Smad2. Expression of the murine EGF-CFC gene cripto rescues oep mutants. These results suggest a conserved role for EGF-CFC proteins as essential extracellular cofactors for Nodal signaling during vertebrate development.     

Development of chick axial mesoderm: specification of prechordal mesoderm by anterior endoderm-derived TGFbeta family signalling

Two populations of axial mesoderm cells can be recognised in the chick embryo, posterior notochord and anterior prechordal mesoderm. We have examined the cellular and molecular events that govern the specification of prechordal mesoderm. We report that notochord and prechordal mesoderm cells are intermingled and share expression of many markers as they initially extend out of Hensen's node. In vitro culture studies, together with in vivo grafting experiments, reveal that early extending axial mesoderm cells are labile and that their character may be defined subsequently through signals that derive from anterior endodermal tissues. Anterior endoderm elicits aspects of prechordal mesoderm identity in extending axial mesoderm by repressing notochord characteristics, briefly maintaining gsc expression and inducing BMP7 expression. Together these experiments suggest that, in vivo, signalling by anterior endoderm may determine the extent of prechordal mesoderm. The transforming growth factor (beta) (TGFbeta) superfamily members BMP2, BMP4, BMP7 and activin, all of which are transiently expressed in anterior endoderm mimic distinct aspects of its patterning actions. Together our results suggest that anterior endoderm-derived TGFbetas may specify prechordal mesoderm character in chick axial mesoderm.     

On the role of fibronectin during the compaction stage of somitogenesis in the chick embryo

During the early stages of somitogenesis in the chick embryo the presomitic cells in the segmental plate undergo compaction. The aggregation of segmental plate cells is stimulated by fibronectin. The stimulation of segmental plate cells to aggregate and undergo compaction can be effected in isolated segmental plate cells, in isolated segmental plates, and in intact embryos removed from the yolk. The fact that the segmental plate cells react with greater vigor to cellular fibronectin than to plasma fibronectin suggests a specific molecular mechanism in the initiation of somitogenesis.     

Structure of the zebrafish fasciclin I-related extracellular matrix protein (betaig-h3) and its characteristic expression during embryogenesis

betaig-h3, which is structurally related to the insect fasciclin I, is assumed to act as a cell adhesion molecule through binding to cell-surface integrins. In this study, we obtained cDNA clones for the zebrafish orthologue of betaig-h3 and examined the expression of the gene (betaig-h3) in zebrafish embryos using in situ hybridization. Expression is first seen at the bud stage in the presomitic mesoderm. Throughout the somitogenesis stage, betaig-h3 is expressed in all the segmented somites, as well as in the presomitic mesoderm (S0 and S-I). High expression is observed in the dorsolateral part of the somite until the mid-somitogenesis stage. At late somitogenesis stages, the betaig-h3 expression in the dorsolateral somite fades away, while expression is upregulated in the ventromedial part of the somite that corresponds to the sclerotome. In embryos after completion of somitogenesis and fry after hatching, betaig-h3 continues to be expressed in the sclerotome. In addition, new expression starts in the mesenchyme cells in the head, pharyngeal arches, and pectoral fins. In the embryonic brain, expression is observed along the anterior and postoptic commissures, as well as along the optic nerve.     

The words of the regulatory code are arranged in a variable manner in highly conserved enhancers

The mammillary body, a ventral specialization of the caudal hypothalamus, lies close to the transition between epichordal and prechordal parts of the forebrain (Puelles and Rubenstein, 2003). This report examines its presumed causal connection with either prechordal or notochordal mesodermal induction, as well as the timing of its specification, in the context of early ventral forebrain patterning. It was recently found that the ephrin receptor gene EphA7 is selectively expressed in the mammillary pouch from early stages of development (HH14: García-Calero et al., 2006). We used mammillary EphA7 expression as well as ventral hypothalamic expression of the gene markers Nkx2.1 and Shh to analyze experimental effects on mammillary specification and morphogenesis after axial mesoderm ablation at stages HH4+ to HH6. Progressively delayed ablation of the prechordal plate revealed its sequential implication in molecular specification of the entire ventral forebrain, including the mammillary and tuberal regions of the hypothalamus. We observed differential contact requirements for induction by the prechordal plate of all the forebrain regions expressing Shh and Nkx2.1, including distant subpallial ones. In contrast, ablation of the anterior notochordal tip at these stages did not elicit significant patterning changes, particularly no effects on mammillary EphA7 expression or mammillary pouch development.     

Sphingosine-1-phosphate receptors regulate individual cell behaviours underlying the directed migration of prechordal plate progenitor cells during zebrafish gastrulation

During vertebrate gastrulation, cells forming the prechordal plate undergo directed migration as a cohesive cluster. Recent studies revealed that E-cadherin-mediated coherence between these cells plays an important role in effective anterior migration, and that platelet-derived growth factor (Pdgf) appears to act as a guidance cue in this process. However, the mechanisms underlying this process at the individual cell level remain poorly understood. We have identified miles apart (mil) as a suppressor of defective anterior migration of the prospective prechordal plate in silberblick (slb)/wnt11 mutant embryos, in which E-cadherin-mediated coherence of cell movement is reduced. mil encodes Edg5, a sphingosine-1-phosphate (S1P) receptor belonging to a family of five G-protein-coupled receptors (S1PRs). S1P is a lipid signalling molecule that has been implicated in regulating cytoskeletal rearrangements, cell motility and cell adhesion in a variety of cell types. We examined the roles of Mil in anterior migration of prechordal plate progenitor cells and found that, in slb embryos injected with mil-MO, cells migrate with increased motility but decreased directionality, without restoring the coherence of cell migration. This indicates that prechordal plate progenitor cells can migrate effectively as individuals, as well as in a coherent cluster of cells. Moreover, we demonstrate that Mil regulates cell motility and polarisation through Pdgf and its intracellular effecter PI3K, but modulates cell coherence independently of the Pdgf/PI3K pathway, thus co-ordinating cell motility and coherence. These results suggest that the net migration of prechordal plate progenitors is determined by different parameters, including motility, persistence and coherence.     

Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse.

BACKGROUND: The process of somitogenesis can be divided into three major events: the prepatterning of the mesoderm; the formation of boundaries between the prospective somites; and the cellular differentiation of the somites. Expression and functional studies have demonstrated the involvement of the murine Notch pathway in somitogenesis, although its precise role in this process is not yet well understood. We examined the effect of mutations in the Notch pathway elements Delta like 1 (Dll1), Notch1 and RBPJkappa on genes expressed in the presomitic mesoderm (PSM) and have defined the spatial relationships of Notch pathway gene expression in this region. RESULTS: We have shown that expression of Notch pathway genes in the PSM overlaps in the region where the boundary between the posterior and anterior halves of two consecutive somites will form. The Dll1, Notch1 and RBPJkappa mutations disrupt the expression of Lunatic fringe (L-fng), Jagged1, Mesp1, Mesp2 and Hes5 in the PSM. Furthermore, expression of EphA4, mCer 1 and uncx4.1, markers for the anterior-posterior subdivisions of the somites, is down-regulated to different extents in Notch pathway mutants, indicating a global alteration of pattern in the PSM. CONCLUSIONS: We propose a model for the mechanism of somite border formation in which the activity of Notch in the PSM is restricted by L-fng to a boundary-forming territory in the posterior half of the prospective somite. In this region, Notch function activates a set of genes that are involved in boundary formation and anterior-posterior somite identity.     

Developmental expression of zebrafish emx1 during early embryogenesis

Emx family homeobox genes, Emx1 and Emx2, play an essential role in rostral brain development in mammalian embryos. Here we report a zebrafish emx family gene, emx1, which is more similar to the mouse Emx1 gene than the previously reported zebrafish emx1 gene; we propose to rename that gene emx3. The expression of emx1 is first detected around the 10-somite stage in the pineal gland (epiphysis) primodium in the developing anterior brain and in the pronephric primodium within the intermediate mesoderm. emx1 expression in the epiphysis has not been reported in other species. Expression in the epiphysis is suppressed at 23 h post-fertilization (hpf) in the floating head (flh) mutant, in which development of the epiphysis is impaired. Subsequently, emx1 is expressed in the telencephalon, as reported in mammals, and can be detected in the olfactory placode and in a small group of cells in the forebrain at 25 hpf. In the mesoderm, emx1 expression is gradually concentrated in the posterior pronephric duct during somitogenesis, and becomes expressed predominantly in the urogenital opening at 25 hpf. Thus, emx1 displays a unique expression pattern that is distinct from the patterns of emx2 and emx3.