The Midbrain–Hindbrain Boundary Organizer Muriel Rhinn and Michael Brand*

34 The midbrain–hindbrain boundary organizer Muriel Rhinn and Michael Brand* Cell fate in the cephalic neural primordium is controlled by an tissue is transplanted into the caudal forebrain of chick organizer located at the midbrain–hindbrain boundary. Studies embryos, the surrounding host tissue switches fate and in chick, mouse and zebrafish converge to show that mutually adopts an isthmic or midbrain character [11,12]; in the repressive interactions between homeodomain transcription rhombencephalon, MHB tissue induces cerebellar fate factors of the Otx and Gbx class position this organizer in the [13]. These experiments suggested that this tissue also neural primordium. Once positioned, independent signaling acts as an organizing center in its normal location at the pathways converge in their activity to drive organizer function. MHB. This review focuses on recent progress in under- Fibroblast growth factors secreted from the organizer are standing how the midbrain–hindbrain boundary organizer necessary for, and sufficient to mimic, organizer activity in develops and functions. patterning the midbrain and anterior hindbrain, and are tightly controlled by feedback inhibition. Several genes, encoding either transcription factors (Engrailed [En], Pax, Otx and Gbx families) or secreted pro- Addresses teins (Wnt and Fgf [fibroblast growth factor] families), are Max Planck Institute for Molecular Cell Biology and Genetics, expressed within the midbrain–hindbrain territory at early Pfotenhauer Strasse 108, 01307 Dresden, Germany embryonic stages (Figure 1). Several groups have generat- *e-mail: [email protected] ed mutations in these genes in mice through gene Correspondence: Michael Brand targeting [9,10]. Mutagenesis screens in zebrafish have Current Opinion in Neurobiology 2001, 11:34–42 yielded acerebellar (ace), a probable null-allele of fgf8, an 0959-4388/01/$ — see front matter allelic series of no isthmus (noi) alleles in the pax2.1 gene © 2001 Elsevier Science Ltd. All rights reserved. [14–16], and several mutants in which molecular identifi- cation is ongoing. The different mutants lack MHB Abbreviations ace acerebellar structures and/or neighboring brain territories to varying ANR anterior neural ridge degrees, as listed in Table 1. From the mutant analysis, E embryonic day several regulatory steps are distinguished in MHB devel- FGF fibroblast growth factor opment. During the establishment phase, a crucial first MHB midbrain–hindbrain boundary step is the subdivision into an Otx2- and a Gbx2-expressing noi no isthmus WT wild-type domain (see below). At this interface between Otx2 and Gbx2, at least three signaling pathways become activated independently of each other, as monitored by the expres- Introduction sion of the wnt1, pax2.1 and fgf8 genes (Figure 2a) [15,16]. The initial subdivision of the neural plate, or regionaliza- Establishment is followed by the maintenance phase, dur- tion, is the first step towards generating cellular diversity in ing which expression of the above genes comes to depend the vertebrate brain. The subdivision is reflected by gene on each other. Perturbation of any one gene disrupts the expression in restricted domains along the length of the continued development of the MHB. During this period, neural primordium. As development proceeds, this rough Fgf8 expression is activated at the MHB, thus probably subdivision is further refined within each region, ultimate- endowing these cells with organizing capacity (Figure 2b). ly generating the multitude of cell types in the central nervous system (CNS). Both vertical signals from the The Otx–Gbx interface and positioning of the mesoderm to the overlying ectoderm [1] and planar signals isthmic organizer — or how much of a fly wing travelling in the plane of the ectodermal epithelium are is the MHB? thought to be involved in generating cell diversity [2–4]. The establishment of organizing centers is thought to require the prior specification of two distinct, adjacent cell popula- Patterning of the neural primordium also involves neu- tions. Local cellular interactions then result in the production roepithelial organizers — special groups of cells that of molecules with longer-range signaling properties [17]. This produce secreted molecules and thus control the cell fate phenomenon has been studied extensively, for example, at of the surrounding cells. The two best-studied organizers the anterior–posterior compartment boundary of the fly wing. are the anterior neural ridge (ANR, or row 1 [the first row How are the two cell populations that generate the MHB of cells in the zebrafish neural plate]) acting on the fore- organizer defined? During normal CNS development, one of brain neural plate [5,6,7•]), and the midbrain–hindbrain the earliest events is the subdivision into an anterior Otx2- boundary organizer (MHB organizer, or isthmic organizer) positive and a posterior Gbx2-positive domain. During late acting on the midbrain and hindbrain primordium [8–10]. gastrulation/early neural plate stages, Otx2 is expressed from the anterior limit of the neural plate to a posterior border at The MHB organizer was initially identified through trans- the presumptive MHB and Gbx2 is expressed in a comple- plantation experiments in chick embryos. When MHB mentary fashion in the posterior embryo [18]. Subsequently, The midbrain–hindbrain boundary organizer Rhinn and Brand 35 Figure 1 Comparison of the onset of expression of the 50% References different genes associated with Zebrafish Stage 60% 80% 90% tb 1s 3s 5s 7s 14s 24h midbrain–hindbrain organizing activity in three otx2 [77] different species: mouse, zebrafish and chick. gbx1 (a) The mRNA expression patterns of the gbx2 (a) different genes (Otx2, Gbx, Fgf8, Wnt1, En fgf8 [16] and Pax) are shown schematically on the wnt1 basis of the results of in situ hybridization [15,16] eng1 analyses. (a) M Brand, unpublished data. [15,16] eng2 [15,16] eng3 [15,16] eng4 [15,16] pax2.1 [15,75] pax2.2 [15,75] pax5 [15,75] pax8 [15,75] Mouse Stage E6.5 E7.5 E7.75 HDF 1s 3s 5s 7s 14s 9.5 dpc Otx2 [78,79] Gbx2 [18,80] Fgf8 [20] Wnt1 [19] En1 [19] En2 [19] Pax2 [19] Pax5 [19] Pax8 [19] Chick Stage HH4 HH5 HH6 HH7 HH8+ HH9 HH12 Otx2 [81] Gbx2 [82] Fgf8 [41•] Wnt1 [41•] En1 [83] En2 [83] Pax2 [41•,84] Pax5 [85] Pax8 Not determined Current Opinion in Neurobiology Pax2 is activated, followed by En1, Wnt1 [19] and Fgf8 MHB organizer with it, are shifted anteriorly at early somite [16,20,21]. These genes are activated around the Otx2–Gbx2 stages. Such embryos form neither midbrain nor caudal fore- interface, consistent with the notion that the region where brain, and the anterior hindbrain is expanded rostrally [31]. Otx2 and Gbx2 abut demarcates the primordium of the MHB. Conversely, Gbx2-null mutants show a failure of anterior Furthermore, the MHB has the ability to regenerate after its hindbrain development and display a caudal expansion of removal, suggesting that it is normally generated and/or main- the midbrain and of Otx2, Wnt1 and Fgf8 expression, appar- tained by cell–cell interactions between Otx2- and ently due to a respecification of the hindbrain at early Gbx2-expressing neuroepithelial cells [22,23••]. In addition, somite stages (six somites) [18,32•]. transplantations, co-cultures and electroporation experiments show that the confrontation of Otx2- and Gbx2-expressing ter- Evidence from misexpression experiments is complemen- ritories activates expression of Fgf8, a key mediator of the tary to that of the loss-of-function studies (Figure 3). When MHB organizing activity [23••,24,25••,26]. Otx2 expression is forced in a more caudal position using an Otx2 transgene driven by an En1 promoter, Gbx2 expres- The above data suggested that creating the Otx2–Gbx2 bor- sion is repressed and the MHB is shifted posteriorly [33•]. der in the right place is important to position the MHB Conversely, ectopic expression of Gbx2 in the caudal mid- organizer, and genetic analysis of Otx2 and Gbx2 in mice pro- brain, driven by a Wnt1–promoter–Gbx2 transgene, vides evidence for this (Figure 3). Otx2-null mutants lack represses Otx2 and shifts the induction of MHB markers to the brain rostral to rhombomere 3 ([27–29]; for a review, see the level of the newly created interface; surprisingly, this [30]). Furthermore, in mutants with a reduced copy number shift appears to be only transient [32•]. These results of Otx genes, the caudal limit of Otx2 expression, and the together suggest that Gbx2 directly or indirectly represses 36 Development Table 1 Phenotypes of embryos carrying a mutation in genes expressed at the MHB. Gene Species MHB mutant phenotype References Otx1 Mouse Homozygous Otx1 mutant adult mice have cortical defects, an abnormal midbrain and [30,31] abnormal cerebellar foliation. Cooperates with Otx1 in MHB development; double mutants show an increase in strength of the embryonic MHB phenotype. Otx2 Mouse Homozygous Otx2 mutant embryos lack the brain rostral to hindbrain rhombomere 3. [27–29,31,34,35,48] Cooperates with Otx1 in MHB development. In chimeric embryos that have only OTX protein in the visceral endoderm, the forebrain and midbrain induction is rescued. Absence of OTX protein in the neuroectoderm leads to incorrect regionalization. Gbx2 Mouse Gbx2 mutant embryos lack anterior hindbrain and show a caudal expansion of the posterior [18,32•] midbrain. The Otx2 expression domain is expanded posteriorly. Consequently, Wnt1 and Fgf8 expression domains are also shifted caudally. Pax2 Mouse The effect of the Pax2 mutation is influenced by the genetic background of the mouse strain [86,87,90] analyzed, ranging from deletion of the posterior midbrain and cerebellum or exencephaly to almost normal development of these structures. Pax2.1 (noi) Zebrafish No isthmus (noi) mutants lack the midbrain, MHB and cerebellum. eng3 activation is completely [14,15,75] and eng2 is strongly dependent on noi function. In contrast, onset of wnt1 and fgf8 occurs normally. Pax5 Mouse Pax5 mutant embryos show defects in the inferior colliculi and anterior cerebellum.

Load more