FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression

Beads containing recombinant FGF8 (FGF8-beads) were implanted in the prospective caudal diencephalon or midbrain of chick embryos at stages 9-12. This induced the neuroepithelium rostral and caudal to the FGF8-bead to form two ectopic, mirror-image midbrains. Furthermore, cells in direct contact with the bead formed an outgrowth that protruded laterally from the neural tube. Tissue within such lateral outgrowths developed proximally into isthmic nuclei and distally into a cerebellum-like structure. These morphogenetic effects were apparently due to FGF8-mediated changes in gene expression in the vicinity of the bead, including a repressive effect on Otx2 and an inductive effect on En1, Fgf8 and Wnt1 expression. The ectopic Fgf8 and Wnt1 expression domains formed nearly complete concentric rings around the FGF8-bead, with the Wnt1 ring outermost. These observations suggest that FGF8 induces the formation of a ring-like ectopic signaling center (organizer) in the lateral wall of the brain, similar to the one that normally encircles the neural tube at the isthmic constriction, which is located at the boundary between the prospective midbrain and hindbrain. This ectopic isthmic organizer apparently sends long-range patterning signals both rostrally and caudally, resulting in the development of the two ectopic midbrains. Interestingly, our data suggest that these inductive signals spread readily in a caudal direction, but are inhibited from spreading rostrally across diencephalic neuromere boundaries. These results provide insights into the mechanism by which FGF8 induces an ectopic organizer and suggest that a negative feedback loop between Fgf8 and Otx2 plays a key role in patterning the midbrain and anterior hindbrain.     

Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the isthmic organizer

The development of the vertebrate brain depends on the formation of local organizing centres within the neural tube that express secreted signals that refine local neural progenitor identity. The isthmic organizer (IsO) forms at the isthmic constriction and is required for the growth and ordered development of mesencephalic and metencephalic structures. The formation of the IsO, which is characterized by the generation of a complex pattern of cells at the midbrain-hindbrain boundary, has been described in detail. However, when neural plate cells are initially instructed to form the IsO, the molecular nature of the inductive signals remain poorly defined. We now provide evidence that convergent Wnt and FGF signaling at the gastrula stage are required to generate the complex polarized pattern of cells characteristic of the IsO, and that Wnt and FGF signals in combination are sufficient to reconstruct, in na?ve forebrain cells, an IsO-like structure that exhibits an organizing activity that mimics the endogenous IsO when transplanted into the diencephalon of chick embryos.     

Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB.     

Embryogenesis and expression profiles of charon and nodal-pathway genes in sinistral (Paralichthys olivaceus) and dextral (Verasper variegatus) flounders

Although it is well known that flounder form external asymmetry by migration of one eye at metamorphosis, the control system that forms this asymmetry is unknown. To help elucidate this mechanism, we here describe the embryogenesis and expression profiles of the Nodal-pathway genes in the Japanese flounder, Paralichthys olivaceus. We also perform a comparative study of the laterality of the expression of these genes in sinistral (P. olivaceus) and dextral (Verasper variegatus) flounders. In P. olivaceus, Kupffer's vesicle forms at the 2-somite stage, after which left-sided expression of spaw starts at the 8-somite stage. Left-sided expression of pitx2 occurs in the gut field at the 15-somite to high-pec stages, in the heart field at the 21-somite stage, and in the dorsal diencephalon at the 27-somite to high-pec stages. In response to left-sided pitx2 expression, the heart, gut, and diencephalon begin asymmetric organogenesis at the pharyngula (heart) and the long-pec (gut and diencephalon) stages, whereas the eyes do not show signs of asymmetry at these stages. In both sinistral and dextral flounders, the Nodal-pathway genes are expressed at the left side of the dorsal diencephalon and left lateral-plate mesoderm. Considering these data together with our previous finding that reversal of eye laterality occurs to some extent in the P. olivaceus mutant reversed, in which embryonic pitx2 expression is randomized, we propose that although the Nodal pathway seems to function to fix eye laterality, embryonic expression of these genes does not act as a direct positional cue for eye laterality.     

Cephalic neurulation and optic vesicle formation in the early mouse embryo.

The overall pattern of cephalic neurulation and the concomitant early development of the optic vesicles in mouse embryos were examined by scanning electron microscopy. Paraffin-sectioned specimens were also examined. The overall pattern of closure of the cephalic neural folds accords well with earlier observations of this process. The earliest indication of optic placode formation was seen in histological sections of embryos at the 4-somite stage, while optic pit formation was first observed at the 5- to 6-somite stage. The upper halves of the optic vesicles were formed in 10- to 15-somite embryos by the fusion of the neural folds at the junction between the mesencephalon and prosencephalon, while closure of the lower halves was associated with the closure of the rostral neuropore, and was usually completed by about the 20-somite stage. By the 25- to 30-somite stage, a rapid increase in the volume of the forebrain was observed, so that the optic vesicles were displaced laterally. An overall increase in the volume of the optic vesicles and decrease in the diameter of the optic stalks were also observed at this time. This account of cephalic neurulation and optic organogenesis provides useful baseline data relevant to the study of the normal early development of the mouse. A comparison is made between similar events in the rat, the hamster, and the human embryo.     

Patterning the zebrafish diencephalon by the conserved zinc-finger protein Fezl

The forebrain constitutes the most anterior part of the central nervous system, and is functionally crucial and structurally conserved in all vertebrates. It includes the dorsally positioned telencephalon and eyes, the ventrally positioned hypothalamus, and the more caudally located diencephalon [from rostral to caudal: the prethalamus, the zona limitans intrathalamica (ZLI), the thalamus and the pretectum]. Although antagonizing Wnt proteins are known to establish the identity of the telencephalon and eyes, it is unclear how various subdivisions are established within the diencephalon--a complex integration center and relay station of the vertebrate brain. The conserved forebrain-specific zinc-finger-containing protein Fezl plays a crucial role in regulating neuronal differentiation in the vertebrate forebrain. Here, we report a new and essential role of zebrafish Fezl in establishing regional subdivisions within the diencephalon. First, reduced activity of fezl results in a deficit of the prethalamus and a corresponding expansion of the ZLI. Second, Gal4-UAS-mediated fezl overexpression in late gastrula is capable of expanding the prethalamus telencephalon and hypothalamus at the expense of the ZLI and other fore- and/or mid-brain regions. Such altered brain regionalization is preceded by the early downregulation of wnt expression in the prospective diencephalon. Finally, fezl overexpression is able to restore the anterior forebrain and downregulate wnt expression in Headless- and/or Tcf3 (also known as Tcf7l1a)-deficient embryos. Our findings reveal that Fezl is crucial for establishing regional subdivisions within the diencephalon and may also play a role in the development of the telencephalon and hypothalamus.     

Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the anterior neuroectoderm for normal anteroposterior (AP) patterning. We have previously identified a zebrafish frizzled8a (fz8a) gene expressed in the presumptive anterior neuroectoderm as well as prechordal plate at the late gastrula stage. We have investigated the role of Fz8a-mediated Wnt8b signalling in anterior brain patterning in zebrafish. We show that in zebrafish embryos: (1) Wnt signalling has at least two different stage-specific posteriorizing activities in the anterior neuroectoderm, one before mid-gastrulation and the other at late gastrulation; (2) Fz8a plays an important role in mediating anterior brain patterning; (3) Wnt8b and Fz8a can functionally interact to transmit posteriorizing signals that determine the fate of the posterior diencephalon and midbrain in late gastrula embryos; and (4) Wnt8b can suppress fz8a expression in the anterior neuroectoderm and potentially affect the level and/or range of Wnt signalling. In conclusion, we suggest that a gradient of Fz8a-mediated Wnt8b signalling may play crucial role in patterning the posterior diencephalon and midbrain regions in the late gastrula.     

Rotation of the tectal primordium reveals plasticity of target recognition in retinotectal projection

Retinotectal projection is precisely organized in a retinotopic manner. In normal projection, temporal retinal axons project to the rostral part of the tectum, and nasal axons to the caudal part of the tectum. The two-dimensional relationship between the retina and the tectum offers a useful experimental system for analysis of neuronal target recognition. We carried out rotation of the tectal primordium in birds at an early stage of development, around the 10-somite stage, to achieve a better understanding of the characteristics of target recognition, especially the rostrocaudal specificity of the tectum. Our results showed that temporal retinal axons projected to the rostral part of the rotated tectum, which was originally caudal, and that nasal axons projected to the caudal part of the rotated tectum, which was originally rostral. Therefore, the tectum that had been rotated at the 10-somite stage received normal topographic projection from the retinal ganglion cells. Rostrocaudal specificity of the tectum for target recognition is not determined by the 10-somite stage and is acquired through interactions between the tectal primordium and its surrounding structures.     

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.     

The midbrain-hindbrain boundary genetic cascade is activated ectopically in the diencephalon in response to the widespread expression of one of its components, the medaka gene Ol-eng2.

In vertebrates, the engrailed genes are expressed at early neurula stage in a narrow stripe encompassing the midbrain-hindbrain boundary (MHB), a region from which a peculiar structure, the isthmus, is formed. Knock-out experiments in mice demonstrated that these genes are essential for the development of this structure and of its derivatives. In contrast, little is known about the effect of an overexpression of engrailed genes in vertebrate development. Here we report the isolation of Ol-eng2, a medaka fish (Oryzias latipes) engrailed gene. We have monitored the effects of its widespread expression following mRNA injections in 1- and 2-cell medaka and Xenopus embryos. We found that the ectopic expression of Ol-eng2 predominantly results in an altered development of the anterior brain, including an inhibition of optic vesicle formation. No change in the patterns of mesencephalic and telencephalic markers were observed. In contrast, expressions of markers of the diencephalon were strongly repressed in injected embryos. Furthermore, the endogenous Ol-eng2, Pax2, Wnt1 and Fgf8, which are essential components of the MHB genetic cascade, were ectopically expressed in this region. Therefore, we propose that Ol-eng2 induces de novo formation of an isthmus-like structure, which correlates with the development of ectopic midbrain structures, including optic tectum. A competence of the diencephalon to change to a midbrain fate has been demonstrated in isthmic graft experiments. Our data demonstrate that this change can be mimicked by ectopic engrailed expression alone.     

Msx1 is required for dorsal diencephalon patterning

The dorsal midline of the neural tube has recently emerged as a major signaling center for dorsoventral patterning. Msx genes are expressed at the dorsal midline, although their function at this site remains unknown. Using Msx1(nlacZ) mutant mice, we show that the normal expression domain of Msx1 is interrupted in the pretectum of mutant embryos. Morphological and gene expression data further indicate that a functional midline is not maintained along the whole prosomere 1 in Msx1 mutant mice. This results in the downregulation of genes expressed laterally to the midline in prosomere 1, confirming the importance of the midline as a signaling center. Wnt1 is essential for dorsoventral patterning of the neural tube. In the Msx1 mutant, Wnt1 is downregulated before the midline disappears, suggesting that its expression depends on Msx1. Furthermore, electroporation in the chick embryo demonstrates that Msx1 can induce Wnt1 expression in the diencephalon neuroepithelium and in the lateral ectoderm. In double Msx1/Msx2 mutants, Wnt1 expression is completely abolished at the dorsal midline of the diencephalon and rostral mesencephalon. This indicates that Msx genes may regulate Wnt1 expression at the dorsal midline of the neural tube. Based on these results, we propose a model in which Msx genes are intermediary between Bmp and Wnt at this site.     

Expression of en in the Prosencephalon Heterotopically Transplanted into the Mesencephalon

The alar plate of the prosencephalon differentiates into a tectum-like structure when transplanted and integrated into the mesencephalon around 10-somite stage. Homeobox containing gene, en , is expressed in the mesencephalon with caudorostral gradient from 7-somite stage. Here expression of en in the prosencephalic transplant in the mesencephalon was studied immunohistochemically with anti- en antibody. Transplantation was carried out between quail and chick. It was shown that the transplant was stained with anti- en antibody at the caudal part of the mesencephalon. At the rostral part, the transplant did not express en . When the transplant was not contiguous to the caudal part of the host mesencephalon, it did not express en . The present study suggests that the prosencephalic transplant differentiates into the tectum-like structure when it expresses en at the caudal part of the mesencephalon.     

Stage-specific response of the mesenchyme to excess vitamin A in developing rat facial processes

The effects of excess retinol (vitamin A alcohol) on facial process formation were examined in cultured rat embryos. The embryos were explanted at day 11 of gestation (plug day = 0) and cultured for 72 hr in rat serum containing an additional 1 or 10 micrograms/ml retinol. The reduction of outgrowth in the facial processes was observed in 1 microgram/ml retinol-treated embryos, and this type of malformation was found to be more severe in 10 micrograms/ml retinol-treated embryos. Histological findings of 10 micrograms/ml retinol-treated embryos at the 50-somite stage showed that the nasal epithelium was developed but folded. In the mesenchyme, there were necrotic cells. Thymidine incorporation by mesenchymal cells in the facial processes was also determined. At the 50-somite stage, the uptake was decreased to 66.4% of control value at 1 microgram/ml retinol, whereas the addition of the same dose of retinol did not cause the inhibition at the 36-, 40-, and 42-somite stages. The uptake at the 50-somite stage was decreased to 23.0% as a result of the 10 micrograms/ml retinol treatment. These results show that the response of the facial mesenchyme to excess retinol is dependent on the development stage and the critical stage of the facial mesenchyme for excess retinol in cultured rat embryos is the 42-somite stage.     

Muscle development in the tambaqui, an important Amazonian food fish

Eggs of the tambaqui Colossoma macropomum were incubated at 28 and 31) C. Somitogenesis started shortly after the formation of the neural plate and notochord. New somites were added at the rate of one every 13 min at 28) C and one every 11 min at 31) C. Myogenesis started in the most rostral myotomes at the 9-somite stage and proceeded in a caudal direction. Mononuclear myotubes with the morphological characteristic of muscle pioneer cells were observed lateral to the notochord. The majority of myotubes were formed from the fusion of 3–6 spindle-shaped myoblasts. Myofibril synthesis started soon after cell fusion at the periphery of myotubes. Close membrane contacts and 'gap'-type junctions were observed between myotubes, immature muscle fibres and at the inter-somite boundary, suggesting that the cells were electrically coupled. Embryos exhibited rhythmic movements at the 20-somite stage, and hatched at the 29–30-somite stage 15–18 h post-fertilisation (PFT) at 28° C and 11 h PFT at 31° C. Larvae hatched at a comparatively early stage of development prior to the completion of somitogenesis and the formation of eye pigment, pectoral fins and jaws. The myotomes comprised a single superficial layer of well-differentiated muscle fibres which contained abundant mitochondria, overlying an inner core of myotubes (presumptive white muscle layer). Differentiation and growth during the larval stages was extremely rapid, and the juvenile stage was reached after little more than 6 days at 28° C.