Role of Lmx1b and Wnt1 in mesencephalon and metencephalon development

The isthmus is the organizing center for the tectum and cerebellum. Fgf8 and Wnt1 are secreted molecules expressed around the isthmus. The function of Fgf8 has been well analyzed, and now accepted as the most important organizing signal. Involvement of Wnt1 in the isthmic organizing activity was suggested by analysis of Wnt1 knockout mice. But its role in isthmic organizing activity is still obscure. Recently, it has been shown that Lmx1b is expressed in the isthmic region and that it may occupy higher hierarchical position in the gene expression cascade in the isthmus. We have carried out misexpression experiment of Lmx1b and Wnt1, and considered their role in the isthmic organizing activity. Lmx1b or Wnt1 misexpression caused expansion of the tectum and cerebellum. Fgf8 was repressed in a cells that misexpress Lmx1b, but Fgf8 expression was induced around Lmx1b-misexpressing cells. As Lmx1b induced Wnt1 and Wnt1 induced Fgf8 expression in turn, Wnt1 may be involved in non cell-autonomous induction of Fgf8 expression by Lmx1b. Wnt1 could not induce Lmx1b expression so that Lmx1b may be put at the higher hierarchical position than Wnt1 in gene expression cascade in the isthmus. We have examined the relationship among isthmus related genes, and discuss the mechanism of the formation and maintenance of isthmic organizing activity.     

Inductive signal and tissue responsiveness defining the tectum and the cerebellum

The mes/metencephalic boundary (isthmus) has an organizing activity for mesencephalon and metencephalon. The candidate signaling molecule is Fgf8 whose mRNA is localized in the region where the cerebellum differentiates. Responding to this signal, the cerebellum differentiates in the metencephalon and the tectum differentiates in the mesencephalon. Based on the assumption that strong Fgf8 signal induces the cerebellum and that the Fgf8b signal is stronger than that of Fgf8a, we carried out experiments to misexpress Fgf8b and Fgf8a in chick embryos. Fgf8a did not affect the expression pattern of Otx2, Gbx2 or Irx2. En2 expression was upregulated in the mesencephalon and in the diencephalon by Fgf8a. Consequently, Fgf8a misexpression resulted in the transformation of the presumptive diencephalon to the fate of the mesencephalon. In contrast, Fgf8b repressed Otx2 expression, but upregulated Gbx2 and Irx2 expression in the mesencephalon. As a result, Fgf8b completely changed the fate of the mesencephalic alar plate to cerebellum. Quantitative analysis showed that Fgf8b signal is 100 times stronger than Fgf8a signal. Co-transfection of Fgf8b with Otx2 indicates that Otx2 is a key molecule in mesencephalic generation. We have shown by RT-PCR that both Fgf8a and Fgf8b are expressed, Fgf8b expression prevailing in the isthmic region. The results all support our working hypothesis that the strong Fgf8 signal induces the neural tissue around the isthmus to differentiate into the cerebellum.     

How does Fgf signaling from the isthmic organizer induce midbrain and cerebellum development?

The mesencephalic/rhombomere 1 border (isthmus) is an organizing center for early development of midbrain and cerebellum. In this review, we summarize recent progress in studies of Fgf signaling in the isthmus and discuss how the isthmus instructs the differentiation of the midbrain versus cerebellum. Fgf8 is shown to play a pivotal role in isthmic organizer activity. Only a strong Fgf signal mediated by Fgf8b activates the Ras-extracellular signal-regulated kinase (ERK) pathway, and this is sufficient to induce cerebellar development. A lower level of signaling transduced by Fgf8a, Fgf17 and Fgf18 induce midbrain development. Numerous feedback loops then maintain appropriate mesencephalon/rhombomere1 and organizer gene expression.     

The Fgf8 signal causes cerebellar differentiation by activating the Ras-ERK signaling pathway

The mes/metencephalic boundary (isthmus) is an organizing center for the optic tectum and cerebellum. Fgf8 is accepted as a crucial organizing signal. Previously, we reported that Fgf8b could induce cerebellum in the mesencephalon, while Fgf8a transformed the presumptive diencephalon into mesencephalon. Since lower doses of Fgf8b exerted similar effects to those of Fgf8a, the type difference could be attributed to the difference in the strength of the signal. It is of great interest to uncover mechanisms of signal transduction pathways downstream of the Fgf8 signal in tectal and cerebellar development, and in this report we have concentrated on the Ras-ERK pathway. In normal embryos, extracellular-signal-regulated kinase (ERK) is activated at the site where Fgf8 mRNA is expressed. Fgf8b activated ERK while Fgf8a or a lower dose of Fgf8b did not activate ERK in the mes/metencephalon. Disruption of the Ras-ERK signaling pathway by a dominant negative form of Ras (RasS17N) changed the fate of the metencephalic alar plate from cerebellum to tectum. RasS17N canceled the effects of Fgf8b, while co-transfection of Fgf8a and RasS17N exerted additive effects. Disruption of Fgf8b, not Fgf8a, by siRNA resulted in posterior extension of the Otx2 expression domain. Our results indicate that the presumptive metencephalon receives a strong Fgf8 signal that activates the Ras-ERK pathway and differentiates into the cerebellum.     

The isthmic organizer signal FGF8 is required for cell survival in the prospective midbrain and cerebellum

Numerous studies have demonstrated that the midbrain and cerebellum develop from a region of the early neural tube comprising two distinct territories known as the mesencephalon (mes) and rostral metencephalon (met; rhombomere 1), respectively. Development of the mes and met is thought to be regulated by molecules produced by a signaling center, termed the isthmic organizer (IsO), which is localized at the boundary between them. FGF8 and WNT1 have been implicated as key components of IsO signaling activity, and previous studies have shown that in Wnt1(-/-) embryos, the mes/met is deleted by the 30 somite stage ( approximately E10) (McMahon, A. P. and Bradley, A. (1990) Cell 62, 1073-1085). We have studied the function of FGF8 in mouse mes/met development using a conditional gene inactivation approach. In our mutant embryos, Fgf8 expression was transiently detected, but then was eliminated in the mes/met by the 10 somite stage ( approximately E8.75). This resulted in a failure to maintain expression of Wnt1 as well as Fgf17, Fgf18, and Gbx2 in the mes/met at early somite stages, and in the absence of the midbrain and cerebellum at E17.5. We show that a major cause of the deletion of these structures is ectopic cell death in the mes/met between the 7 and 30 somite stages. Interestingly, we found that the prospective midbrain was deleted at an earlier stage than the prospective cerebellum. We observed a remarkably similar pattern of cell death in Wnt1 null homozygotes, and also detected ectopic mes/met cell death in En1 null homozygotes. Our data show that Fgf8 is part of a complex gene regulatory network that is essential for cell survival in the mes/met.     

Regulation of isthmic Fgf8 signal by sprouty2

Fgf8 functions as an organizer at the mes/metencephalic boundary (isthmus). We showed that a strong Fgf8 signal activates the Ras-ERK signaling pathway to organize cerebellar differentiation. Sprouty2 is expressed in an overlapping manner to Fgf8, and is induced by Fgf8. Its function, however, is indicated to antagonize Ras-ERK signaling. Here, we show the regulation of Fgf8 signaling in relation to Sprouty2. sprouty2 expression was induced very rapidly by Fgf8b, but interfered with ERK activation. sprouty2 misexpression resulted in a fate change of the presumptive metencephalon to the mesencephalon. Misexpression of a dominant negative form of Sprouty2 augmented ERK activation, and resulted in anterior shift of the posterior border of the tectum. The results indicate that Fgf8 activates the Ras-ERK signaling pathway to differentiate the cerebellum, and that the hyper- or hypo-signaling of this pathway affects the fate of the brain vesicles. Sprouty2 may regulate the Fgf8-Ras-ERK signaling pathway for the proper regionalization of the metencephalon and mesencephalon.     

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.     

FGF8 can activate Gbx2 and transform regions of the rostral mouse brain into a hindbrain fate.

The mid/hindbrain junction region, which expresses Fgf8, can act as an organizer to transform caudal forebrain or hindbrain tissue into midbrain or cerebellar structures, respectively. FGF8-soaked beads placed in the chick forebrain can similarly induce ectopic expression of mid/hindbrain genes and development of midbrain structures (Crossley, P. H., Martinez, S. and Martin, G. R. (1996) Nature 380, 66-68). In contrast, ectopic expression of Fgf8a in the mouse midbrain and caudal forebrain using a Wnt1 regulatory element produced no apparent patterning defects in the embryos examined (Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997) Development 124, 959-969). We show here that FGF8b-soaked beads can not only induce expression of the mid/hindbrain genes En1, En2 and Pax5 in mouse embryonic day 9.5 (E9.5) caudal forebrain explants, but also can induce the hindbrain gene Gbx2 and alter the expression of Wnt1 in both midbrain and caudal forebrain explants. We also show that FGF8b-soaked beads can repress Otx2 in midbrain explants. Furthermore, Wnt1-Fgf8b transgenic embryos in which the same Wnt1 regulatory element is used to express Fgf8b, have ectopic expression of En1, En2, Pax5 and Gbx2 in the dorsal hindbrain and spinal cord at E10.5, as well as exencephaly and abnormal spinal cord morphology. More strikingly, Fgf8b expression in more rostral brain regions appears to transform the midbrain and caudal forebrain into an anterior hindbrain fate through expansion of the Gbx2 domain and repression of Otx2 as early as the 7-somite stage. These findings suggest that normal Fgf8 expression in the anterior hindbrain not only functions to maintain development of the entire mid/hindbrain by regulating genes like En1, En2 and Pax5, but also might function to maintain a metencephalic identity by regulating Gbx2 and Otx2 expression.     

Apc1 is required for maintenance of local brain organizers and dorsal midbrain survival

The tumor suppressor Apc1 is an intracellular antagonist of the Wnt/β-catenin pathway, which is vital for induction and patterning of the early vertebrate brain. However, its role in later brain development is less clear. Here, we examined the mechanisms underlying effects of an Apc1 zygotic-effect mutation on late brain development in zebrafish. Apc1 is required for maintenance of established brain subdivisions and control of local organizers such as the isthmic organizer (IsO). Caudal expansion of Fgf8 from IsO into the cerebellum is accompanied by hyperproliferation and abnormal cerebellar morphogenesis. Loss of apc1 results in reduced proliferation and apoptosis in the dorsal midbrain. Mosaic analysis shows that Apc is required cell-autonomously for maintenance of dorsal midbrain cell fate. The tectal phenotype occurs independently of Fgf8-mediated IsO function and is predominantly caused by stabilization of β-catenin and subsequent hyperactivation of Wnt/β-catenin signalling, which is mainly mediated through LEF1 activity. Chemical activation of the Wnt/β-catenin in wild-type embryos during late brain maintenance stages phenocopies the IsO and tectal phenotypes of the apc mutants. These data demonstrate that Apc1-mediated restriction of Wnt/β-catenin signalling is required for maintenance of local organizers and tectal integrity.     

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.     

Early development of the cerebellum in teleost fishes: a study based on gene expression patterns and histology in the medaka embryo

The cerebellar structures of teleosts are markedly different from those of other vertebrates. The cerebellum continues rostrally into the midbrain ventricle, forming the valvula cerebelli, only in ray-finned fishes among vertebrates. To analyze the ontogenetic processes that underlie this morphological difference, we examined the early development of the cerebellar regions, including the isthmus (mid/hindbrain boundary, MHB), of the medaka (Oryzias latipes), by histology and in-situ hybridization using two gene (wnt1 and fgf8) probes. Isthmic wnt1 was expressed stably in the caudalmost mesencephalic region in the neural tube at all developmental stages examined, defining molecularly the caudal limit of the mesencephalon. The wnt1-positive mesencephalic cells became located rostrally to the isthmic constriction at Iwamatsu's stages 25-26. Isthmic fgf8 expression changed dynamically and became restricted to the rostralmost metencephalic region at stage 24. The rostralmost part (prospective valvula cerebelli) of the fgf8-positive rostral metencephalon protruded rostrally into the midbrain ventricle, bypassing the isthmic constriction, at stages 25-26. Thus, the isthmic constriction shifted caudally with respect to the molecularly defined MHB at stages 25-26. Paired cerebellar primordia were formed from the alar plates of the fgf8-positive rostral metencephalon and the fgf8-negative caudal metencephalon in the medaka neural tube. Our results show that cerebellar development differs between teleosts and murines: both the rostral and caudal metencephalic alar plates develop into the cerebellum in medaka, whereas in the murines only the caudal metencephalic alar plate develops into the cerebellum, and the rostral plate is reduced to a thin membrane.     

Mkp3 is a negative feedback modulator of Fgf8 signaling in the mammalian isthmic organizer

The pivotal mechanisms that govern the correct patterning and regionalization of the distinct areas of the mammalian CNS are driven by key molecules that emanate from the so-called secondary organizers at neural plate and tube stages. FGF8 is the candidate morphogenetic molecule to pattern the mesencephalon and rhombencephalon in the isthmic organizer (IsO). Recognizable relevance has been given to the intracellular pathways by which Fgf8 is regulated and modulated. In chick limb bud development, a dual mitogen-activated protein kinase phosphatase-3 (Mkp3) plays a role as a negative feedback modulator of Fgf8 signaling. We have investigated the role of Mkp3 and its functional relationship with the Fgf8 signaling pathway in the mouse IsO using gene transfer microelectroporation assays and protein-soaked bead experiments. Here, we demonstrate that MKP3 has a negative feedback action on the MAPK/ERK-mediated FGF8 pathway in the mouse neuroepithelium.     

FGF10 can induce Fgf8 expression concomitantly with En1 and R-fng expression in chick limb ectoderm, independent of its dorsoventral specification

The limb bud has a thickened epithelium at the dorsal-ventral boundary, the apical ectodermal ridge (AER), which sustains limb outgrowth and patterning. A secreted molecule fibroblast growth factor (FGF)10 is involved in inducing Fgf8 expression in the prospective AER and mutual interaction between mesenchymal FGF10 and FGF8 in the AER is essential for limb outgrowth. A secreted factor Wnt7a and a homeobox protein Lmx1 are involved in the dorsal patterning of the limb, whereas a homeobox protein Engrailed 1 (En1) is involved in the dorsal-ventral patterning as well as AER formation. Radical fringe (R-fng), a vertebrate homolog of Drosophila fringe was also found to elaborate AER formation in chicks. However, little is known about the molecular interactions between these factors during AER formation. The present study clarified the relationship between FGF10, Wnt7a, Lmx1, R-fng and En1 during limb development using a foil-barrier insertion experiment. It was found that a foil-barrier inserted into the chick prospective wing mesenchyme lateral to the mesonephric duct blocks AER induction. This experiment was expanded by implanting Fgf10-expressing cells lateral to the barrier and examined whether FGF10 could rescue the expression of the limb-patterning genes reported in AER formation. It was found that FGF10 is sufficient to induce Fgf8 expression in the ectoderm of the foil-inserted limb bud, concomitantly with R-fng and En1 expression. However, FGF10 could not rescue the expression of the dorsal marker genes, Wnt7a or Lmx1. Thus, it is suggested that epithelial factors of En1 and R-fng can induce Fgf8 expression in the limb ectoderm in cooperation with a mesenchymal factor FGF10. Some factor(s) other than FGF10, possibly from the paraxial structures medial to the limb mesoderm, is responsible for the initial dorsal-ventral specification of the limb bud.