Neuroepithelial co-expression of Gbx2 and Otx2 precedes Fgf8 expression in the isthmic organizer

The most studied secondary neural organizer is the isthmic organizer, which is localized at the mid-hindbrain transition of the neural tube and controls the anterior hindbrain and midbrain regionalization. Otx2 and Gbx2 expressions are fundamental for positioning the organizer and the establishment of molecular interactions that induce Fgf8. We present here evidences demonstrating that Otx2 and Gbx2 have an overlapping expression in the isthmic region. This area is the transversal domain where expression of Fgf8 is induced. The Fgf8 protein produced in the isthmus stabilizes and up-regulates Gbx2 expression, which, in turn, down-regulates Otx2 expression. The inductive effect of the Gbx2/Otx2 limit keeps Fgf8 expression stable and thus maintains its positive role in the expression of Pax2, En1,2 and Wnt1.     

Fgf8 and Gbx2 induction concomitant with Otx2 repression is correlated with midbrain-hindbrain fate of caudal prosencephalon.

Chick/quail transplantation experiments were performed to analyse possible factors involved in the regionalisation of the midbrain-hindbrain domain. The caudal prosomeres, expressing Otx2, were transplanted at stage HH10 into rostrocaudal levels of the midbrain-hindbrain domain, either straddling the intra-metencephalic constriction (type 1 grafts), or at rostral and medial levels of pro-rhombomere A1 (type 2 and 3 grafts, respectively); thus, in all situations, one border of the graft was in contact with the host Gbx2- and Fgf8-expressing domains. The area containing the graft, recognised by QCPN immunohistochemistry, was first analysed 48 hours after transplantation for Otx2, Gbx2, En2 and Fgf8. Although in all three situations, a large part of the graft maintained Otx2 expression, another part became Otx2 negative and was induced to express Gbx2 and Fgf8. These inductive events occurred exclusively at the interface between the Otx2-positive transplanted domain and the ipsilateral host Gbx2-positive rhombomere 1, creating a new Otx2-Gbx2 boundary within the grafted territory. In type 1 and 2 grafts, the induced Fgf8 domain is in continuity with the host Fgf8 isthmic domain, whereas for type 3 grafts, these two domains are separate. High levels of En2 expression were also induced in the area expressing Gbx2 and Fgf8, and Wnt1 and Pax2 expressions, analysed in type 3 grafts, were induced at the intragraft Otx2-Gbx2 new boundary. Moreover, at later embryonic stages, the graft developed meso-isthmo-cerebellar structures. Thus, gene expressions induced in the grafted prosencephalon not only mimicked the pattern observed in the normal midbrain-hindbrain domain, but is followed by midbrain-hindbrain cytodifferentiation, indicating that not only Fgf8 but also confrontation of Otx2 and Gbx2 may play an essential role during midbrian-hindbrain regionalisation.     

Formation of the boundary between the midbrain and the hindbrain: involvement of Otx2 and Gbx2 genes

We have studied the neuromeric organisation of the mesencephalic-metencephalic (mes-met) territory of the avian neural tube using chick/quail transplantation experiments and analysing the expression of various regulatory genes in chimeric and normal embryos. Homotopic grafts demonstrate the presence of an interneuromeric boundary separating the mesencephalic and cerebellar territories (the mes-met or midbrain/hindbrain boundary). This boundary is characterised from HH10 onwards by the confrontation of the Otx2-Wnt1 and Gbx2-Fgf8 expressing domains, while En2 and Pax2 genes are expressed at both sides of the mes-met boundary. The evolution of the position of the Otx2/Gbx2 boundary with respect to the vesicles and constriction observed within the mes-met domain between stages HH10 and HH20, allows us to redefine the fate map of this region and to propose a new nomenclature for HH10. Transplantation between the prosencephalic neuroepithelium and the mes-met domain shows the possibility of inducing a mes-met phenotype within the two caudal-most prosomeres, preceded by its characteristic genetic cascade. The induction selectively takes place along the boundary between the graft (Otx2 positive) and the host cerebellar territory (expressing high levels of Gbx2); this includes the induction inside the graft of a new Otx2/Gbx2 boundary. Conversely, no induction is ever observed when the graft is confronted to the host Otx2 expressing domain. Although Fgf8 may be involved in the inductive events, our data strongly suggest that confrontation between Otx2 and Gbx2 is essential as an organiser of the mes-met domain.     

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.     

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.     

Otx2 and Gbx2 are required for refinement and not induction of mid-hindbrain gene expression.

Otx2 and Gbx2 are among the earliest genes expressed in the neuroectoderm, dividing it into anterior and posterior domains with a common border that marks the mid-hindbrain junction. Otx2 is required for development of the forebrain and midbrain, and Gbx2 for the anterior hindbrain. Furthermore, opposing interactions between Otx2 and Gbx2 play an important role in positioning the mid-hindbrain boundary, where an organizer forms that regulates midbrain and cerebellum development. We show that the expression domains of Otx2 and Gbx2 are initially established independently of each other at the early headfold stage, and then their expression rapidly becomes interdependent by the late headfold stage. As we demonstrate that the repression of Otx2 by retinoic acid is dependent on an induction of Gbx2 in the anterior brain, molecules other than retinoic acid must regulate the initial expression of Otx2 in vivo. In contrast to previous suggestions that an interaction between Otx2- and Gbx2-expressing cells may be essential for induction of mid-hindbrain organizer factors such as Fgf8, we find that Fgf8 and other essential mid-hindbrain genes are induced in a correct temporal manner in mouse embryos deficient for both Otx2 and Gbx2. However, expression of these genes is abnormally co-localized in a broad anterior region of the neuroectoderm. Finally, we find that by removing Otx2 function, development of rhombomere 3 is rescued in Gbx2(-/-) embryos, showing that Gbx2 plays a permissive, not instructive, role in rhombomere 3 development. Our results provide new insights into induction and maintenance of the mid-hindbrain genetic cascade by showing that a mid-hindbrain competence region is initially established independent of the division of the neuroectoderm into an anterior Otx2-positive domain and posterior Gbx2-positive domain. Furthermore, Otx2 and Gbx2 are required to suppress hindbrain and midbrain development, respectively, and thus allow establishment of the normal spatial domains of Fgf8 and other genes.     

Interaction between Otx2 and Gbx2 defines the organizing center for the optic tectum

Otx2 is expressed in the mesencephalon and prosencephalon, and Gbx2 is expressed in the rhombencephalon around stage 10. Loss-of-function studies of these genes in mice have revealed that Otx2 is indispensable for the development of the anterior brain segment, and that Gbx2 is required for the development of the isthmus. We carried out gain-of-function experiments of these genes in chick embryos with a newly developed gene transfer system, in ovo electroporation. When Otx2 was ectopically expressed caudally beyond the midbrain-hindbrain boundary (MHB), the alar plate of the metencephalon differentiated into the optic tectum instead of differentiating into the cerebellum. On the other hand, when Gbx2 was ectopically expressed at the mesencephalon, the caudal limit of the tectum shifted rostrally. We looked at the effects of misexpression on the isthmus- and tectum-related molecules. Otx2 and Gbx2 interacted to repress each other's expression. Ectopic Otx2 and Gbx2 repressed endogenous expression of Fgf8 in the isthmus, but induced Fgf8 expression at the interface between Otx2 and Gbx2 expression. Thus, it is suggested that interaction between Otx2 and Gbx2 determines the site of Fgf8 expression and the posterior limit of the tectum.     

A Gbx homeobox gene in amphioxus: insights into ancestry of the ANTP class and evolution of the midbrain/hindbrain boundary

In the vertebrate central nervous system (CNS), mutual antagonism between posteriorly expressed Gbx2 and anteriorly expressed Otx2 positions the midbrain/hindbrain boundary (MHB), but does not induce MHB organizer genes such as En, Pax2/5/8 and Wnt1. In the CNS of the cephalochordate amphioxus, Otx is also expressed anteriorly, but En, Pax2/5/8 and Wnt1 are not expressed near the caudal limit of Otx, raising questions about the existence of an MHB organizer in amphioxus. To investigate the evolutionary origins of the MHB, we cloned the single amphioxus Gbx gene. Fluorescence in situ hybridization showed that, as in vertebrates, amphioxus Gbx and the Hox cluster are on the same chromosome. From analysis of linked genes, we argue that during evolution a single ancestral Gbx gene duplicated fourfold in vertebrates, with subsequent loss of two duplicates. Amphioxus Gbx is expressed in all germ layers in the posterior 75% of the embryo, and in the CNS, the Gbx and Otx domains abut at the boundary between the cerebral vesicle (forebrain/midbrain) and the hindbrain. Thus, the genetic machinery to position the MHB was present in the protochordate ancestors of the vertebrates, but is insufficient for induction of organizer genes. Comparison with hemichordates suggests that anterior Otx and posterior Gbx domains were probably overlapping in the ancestral deuterostome and came to abut at the MHB early in the chordate lineage before MHB organizer properties evolved.     

An urbilaterian origin of the tripartite brain: developmental genetic insights from Drosophila

Studies on expression and function of key developmental control genes suggest that the embryonic vertebrate brain has a tripartite ground plan that consists of a forebrain/midbrain, a hindbrain and an intervening midbrain/hindbrain boundary region, which are characterized by the specific expression of the Otx, Hox and Pax2/5/8 genes, respectively. We show that the embryonic brain of the fruitfly Drosophila melanogaster expresses all three sets of homologous genes in a similar tripartite pattern. Thus, a Pax2/5/8 expression domain is located at the interface of brain-specific otd/Otx2 and unpg/Gbx2 expression domains anterior to Hox expression regions. We identify this territory as the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain. Mutational inactivation of otd/Otx2 and unpg/Gbx2 result in the loss or misplacement of the brain-specific expression domains of Pax2/5/8 and Hox genes. In addition, otd/Otx2 and unpg/Gbx2 appear to negatively regulate each other at the interface of their brain-specific expression domains. Our studies demonstrate that the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain displays developmental genetic features similar to those observed for the midbrain/hindbrain boundary region in vertebrate brain development. This suggests that a tripartite organization of the embryonic brain was already established in the last common urbilaterian ancestor of protostomes and deuterostomes.     

Gbx2 and Fgf8 are sequentially required for formation of the midbrain-hindbrain compartment boundary

In vertebrates, the common expression border of two homeobox genes, Otx2 and Gbx2, demarcates the prospective midbrain-hindbrain border (MHB) in the neural plate at the end of gastrulation. The presence of a compartment boundary at the MHB has been demonstrated, but the mechanism and timing of its formation remain unclear. We show by genetic inducible fate mapping using a Gbx2(CreER) knock-in mouse line that descendants of Gbx2(+) cells as early as embryonic day (E) 7.5 do not cross the MHB. Without Gbx2, hindbrain-born cells abnormally populate the entire midbrain, demonstrating that Gbx2 is essential for specifying hindbrain fate. Gbx2(+) and Otx2(+) cells segregate from each other, suggesting that mutually exclusive expression of Otx2 and Gbx2 in midbrain and hindbrain progenitors is responsible for cell sorting in establishing the MHB. The MHB organizer gene Fgf8, which is expressed as a sharp transverse band immediately posterior to the lineage boundary at the MHB, is crucial in maintaining the lineage-restricted boundary after E7.5. Partial deletion of Fgf8 disrupts MHB lineage separation. Activation of FGF pathways has a cell-autonomous effect on cell sorting in midbrain progenitors. Therefore, Fgf8 from the MHB may signal the nearby mesencephalic cells to impart distinct cell surface characteristics or induce local cell-cell signaling, which consequently prevents cell movements across the MHB. Our findings reveal the distinct function of Gbx2 and Fgf8 in a stepwise process in the development of the compartment boundary at the MHB and that Fgf8, in addition to its organizer function, plays a crucial role in maintaining the lineage boundary at the MHB by restricting cell movement.     

The isthmic organizer and brain regionalization

Distinct neural identities are acquired through progressive restriction of developmental potential under the influence of local environmental signals. Evidence for the localization of such morphogenetic signals at specific locations of the developing neural primordium has suggested the concept of "secondary organizer regions", which regulate the identity and regional polarity of neighboring neuroepithelial areas one step further. In recent years, the most studied secondary organizer has been the isthmic organizer, which is localized at the hind-midbrain transition and controls anterior hindbrain and midbrain regionalization. Otx2 and Gbx2 expression is fundamental for positioning the organizer and for the establishment of molecular interactions that induce Fgf8 expression and then, stabilize the autoregulative loop of En1, Wnt1 and Pax2 expression. Temporospatial patterns of such gene expressions are necessary for the correct development of the organizer which, by a planar mechanism of induction, controls the normal development of the rostral hindbrain from r2 to the midbrain-diencephalic boundary. Fgf8 appears as the active diffusible molecule for isthmic morphogenetic activity and has been suggested to be the morphogenetic effector in other inductive activities revealed in other neuroepithelial regions.