Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection.

The organizer at the midbrain-hindbrain boundary (MHB organizer) has been proposed to induce and polarize the midbrain during development. We investigate the requirement for the MHB organizer in acerebellar mutants, which lack a MHB and cerebellum, but retain a tectum, and are mutant for fgf8, a candidate inducer and polarizer. We examine the retinotectal projection in the mutants to assay polarity in the tectum. In mutant tecta, retinal ganglion cell (RGC) axons form overlapping termination fields, especially in the ventral tectum, and along both the anterior-posterior and dorsal-ventral axis of the tectum, consistent with a MHB requirement in generating midbrain polarity. However, polarity is not completely lost in the mutant tecta, in spite of the absence of the MHB. Moreover, graded expression of the ephrin family ligand Ephrin-A5b is eliminated, whereas Ephrin-A2 and Ephrin-A5a expression is leveled in acerebellar mutant tecta, showing that ephrins are differentially affected by the absence of the MHB. Some RGC axons overshoot beyond the mutant tectum, suggesting that the MHB also serves a barrier function for axonal growth. By transplanting whole eye primordia, we show that mapping defects and overshooting largely, but not exclusively, depend on tectal, but not retinal genotype, and thus demonstrate an independent function for Fgf8 in retinal development. The MHB organizer, possibly via Fgf8 itself, is thus required for midbrain polarisation and for restricting axonal growth, but other cell populations may also influence midbrain polarity.     

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.     

Engrailed and Fgf8 act synergistically to maintain the boundary between diencephalon and mesencephalon

Specification of the forebrain, midbrain and hindbrain primordia occurs during gastrulation in response to signals that pattern the gastrula embryo. Following establishment of the primordia, each brain part is thought to develop largely independently from the others under the influence of local organizing centers like the midbrain-hindbrain boundary (MHB, or isthmic) organizer. Mechanisms that maintain the integrity of brain subdivisions at later stages are not yet known. To examine such mechanisms in the anterior neural tube, we have studied the establishment and maintenance of the diencephalic-mesencephalic boundary (DMB). We show that maintenance of the DMB requires both the presence of a specified midbrain and a functional MHB organizer. Expression of pax6.1, a key regulator of forebrain development, is posteriorly suppressed by the Engrailed proteins, Eng2 and Eng3. Mis-expression of eng3 in the forebrain primordium causes downregulation of pax6.1, and forebrain cells correspondingly change their fate and acquire midbrain identity. Conversely, in embryos lacking both eng2 and eng3, the DMB shifts caudally into the midbrain territory. However, a patch of midbrain tissue remains between the forebrain and the hindbrain primordia in such embryos. This suggests that an additional factor maintains midbrain cell fate. We find that Fgf8 is a candidate for this signal, as it is both necessary and sufficient to repress pax6.1 and hence to shift the DMB anteriorly independently of the expression status of eng2/eng3. By examining small cell clones that are unable to receive an Fgf signal, we show that cells in the presumptive midbrain neural plate require an Fgf signal to keep them from following a forebrain fate. Combined loss of both Eng2/Eng3 and Fgf8 leads to complete loss of midbrain identity, resulting in fusion of the forebrain and the hindbrain primordia. Thus, Eng2/Eng3 and Fgf8 are necessary to maintain midbrain identity in the neural plate and thereby position the DMB. This provides an example of a mechanism needed to maintain the subdivision of the anterior neural plate into forebrain and midbrain.     

fgfr3 and regionalization of anterior neural tube in zebrafish

Here we describe the isolation of the zebrafish fgfr3 gene, its structure and chromosomal location. Expression in wild type embryos occurs in the axial mesoderm, the diencephalon, the anterior hindbrain and the anterior spinal cord. In the hindbrain, a differential expression of fgfr3 was detected at several levels of intensity, with the highest expression in the posterior rhombomere 1 that is morphologically distinct from the anterior part, which develops into the cerebellum. Further, analysis of fgfr3 expression in mutants deficient in the formation of the midbrain-hindbrain boundary (MHB), noi(-/-) and ace(-/-), demonstrated that in the absence of Pax2.1 and FGF8 activity, the expression domains of FGFR3 expand into the MHB, tegmentum, cerebellum and optic tectum, which are the affected structures in these mutants.     

FGF regulated gene-expression and neuronal differentiation in the developing midbrain-hindbrain region

The neuroectodermal tissue close to the midbrain-hindbrain boundary (MHB) is an important secondary organizer in the developing neural tube. This so-called isthmic organizer (IsO) secretes signaling molecules, such as fibroblast growth factors (FGFs), which regulate cellular survival, patterning and proliferation in the midbrain and rhombomere 1 (R1) of the hindbrain. We have previously shown that FGF-receptor 1 (FGFR1) is required for the normal development of this brain region in the mouse embryo. Here, we have compared the gene expression profiles of midbrain-R1 tissues from wild-type embryos and conditional Fgfr1 mutants, in which FGFR1 is inactivated in the midbrain and R1. Loss of Fgfr1 results in the downregulation of several genes expressed close to the midbrain-hindbrain boundary and in the disappearance of gene expression gradients in the midbrain and anterior hindbrain. Our screen identified several previously uncharacterized genes which may participate in the development of midbrain-R1 region. Our results also show altered neurogenesis in the midbrain and R1 of the Fgfr1 mutants. Interestingly, the neuronal progenitors in midbrain and R1 show different responses to the loss of signaling through FGFR1.     

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.     

New regulatory interactions and cellular responses in the isthmic organizer region revealed by altering Gbx2 expression

The mouse homeobox gene Gbx2 is first expressed throughout the posterior region of the embryo during gastrulation, and becomes restricted to rhombomeres 1-3 (r1-3) by embryonic day 8.5 (E8.5). Previous studies have shown that r1-3 do not develop in Gbx2 mutants and that there is an early caudal expansion of the midbrain gene Otx2 to the anterior border of r4. Furthermore, expression of Wnt1 and Fgf8, two crucial components of the isthmic organizer, is no longer segregated to adjacent domains in Gbx2 mutants. In this study, we extend the phenotypic analysis of Gbx2 mutants by showing that Gbx2 is not only required for development of r1-3, but also for normal gene expression in r4-6. To determine whether Gbx2 can alter hindbrain development, we generated Hoxb1-Gbx2 (HG) transgenic mice in which Gbx2 is ectopically expressed in r4. We show that Gbx2 is not sufficient to induce r1-3 development in r4. To test whether an Otx2/Gbx2 interface can induce r1-3 development, we introduced the HG transgene onto a Gbx2-null mutant background and recreated a new Otx2/Gbx2 border in the anterior hindbrain. Development of r3, but not r1 and r2, is rescued in Gbx2-/-; HG embryos. In addition, the normal spatial relationship of Wnt1 and Fgf8 is established at the new Otx2/Gbx2 border, demonstrating that an interaction between Otx2 and Gbx2 is sufficient to produce the normal pattern of Wnt1 and Fgf8 expression. However, the expression domains of Fgf8 and Spry1, a downstream target of Fgf8, are greatly reduced in mid/hindbrain junction area of Gbx2-/-; HG embryos and the posterior midbrain is truncated because of abnormal cell death. Interestingly, we show that increased cell death and a partial loss of the midbrain are associated with increased expression of Fgf8 and Spry1 in Gbx2 conditional mutants that lack Gbx2 in r1 after E9.0. These results together suggest that cell survival in the posterior midbrain is positively or negatively regulated by Fgf8, depending on Fgf8 expression level. Our studies provide new insights into the regulatory interactions that maintain isthmic organizer gene expression and the consequences of altered levels of organizer gene expression on cell survival.     

Cloning,expression and relationship of zebrafish gbx1 and gbx2 genes to Fgf signaling

The organizer at the midbrain-hindbrain boundary (MHB) forms at the interface between Otx2 and Gbx2 expressing cell populations, but how these gene expression domains are set up and integrated with the remaining machinery controlling MHB development is unclear. Here we report the isolation, mapping, chromosomal synteny and spatiotemporal expression of gbx1 and gbx2 in zebrafish. We focus in particular on the expression of these genes during development of the midbrain-hindbrain territory. Our results suggest that these genes function in this area in a complex fashion, as evidenced by their highly dynamic expression patterns and relation to Fgf signaling. Analysis of gbx1 and gbx2 expression during formation of the MHB in mutant embryos for pax2.1, fgf8 and pou2 (noi, ace, spg), as well as Fgf-inhibition experiments, show that gbx1 acts upstream of these genes in MHB development. In contrast, gbx2 activation requires ace (fgf8) function, and in the hindbrain primordium, also spg (pou2). We propose that in zebrafish, gbx genes act repeatedly in MHB development, with gbx1 acting during the positioning period of the MHB at gastrula stages, and gbx2 functioning after initial formation of the MHB, from late gastrulation stages onwards. Transplantation studies furthermore reveal that at the gastrula stage, Fgf8 signals from the hindbrain primordium into the underlying mesendoderm. Apart from the general involvement of gbx genes in MHB development reported also in other vertebrates, these results emphasize that early MHB development can be divided into multiple steps with different genetic requirements with respect to gbx gene function and Fgf signaling. Moreover, our results provide an example for switching of a specific gene function of gbx1 versus gbx2 between orthologous genes in zebrafish and mammals.     

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.     

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.     

Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures

The midbrain-hindbrain (MHB) junction has the properties of an organizer that patterns the MHB region early in vertebrate development. Fgf8 is thought to mediate this organizer function. In addition to Fgf8, Fgf17 and Fgf18 are also expressed in the MHB junction. Fgf17 is expressed later and broader than either Fgf8 or Fgf18. Disrupting the Fgf17 gene in the mouse decreased precursor cell proliferation in the medial cerebellar (vermis) anlage after E11.5. Loss of an additional copy of Fgf8 enhanced the phenotype and accelerated its onset, demonstrating that both molecules cooperate to regulate the size of the precursor pool of cells that develop into the cerebellar vermis. However, expression patterns of Wnt1, En2, Pax5 and Otx2 were not altered suggesting that specification and patterning of MHB tissue was not perturbed and that these FGFs are not required to pattern the vermis at this stage of development. The consequence of this developmental defect is a progressive, dose-dependent loss of the most anterior lobe of the vermis in mice lacking Fgf17 and in mice lacking Fgf17 and one copy of Fgf8. Significantly, the differentiation of anterior vermis neuroepithelium was shifted rostrally and medially demonstrating that FGF also regulates the polarized progression of differentiation in the vermis anlage. Finally, this developmental defect results in an ataxic gait in some mice.     

FGFR1 is independently required in both developing mid- and hindbrain for sustained response to isthmic signals

Fibroblast growth factors (FGFs) are signaling molecules of the isthmic organizer, which regulates development of the midbrain and cerebellum. Tissue-specific inactivation of one of the FGF receptor (FGFR) genes, Fgfr1, in the midbrain and rhombomere 1 of the hindbrain of mouse embryos results in deletion of the inferior colliculi in the posterior midbrain and vermis of the cerebellum. Analyses of both midbrain-hindbrain and midbrain-specific Fgfr1 mutants suggest that after establishment of the isthmic organizer, FGFR1 is needed for continued response to the isthmic signals, and that it has direct functions on both sides of the organizer. In addition, FGFR1 appears to modify cell adhesion properties critical for maintaining a coherent organizing center. This may be achieved by regulating expression of specific cell-adhesion molecules at the midbrain-hindbrain border.