Based on histological and anatomical studies, it has beensuggested that cell type is determined by genetic

elements1. Over the past decade, molecules that can conferor modify cell identity have been identified and functionallycharacterized. Signals that can alter cell fate are emitted byan organizer and act on targets in either adjacent tissues(vertical signals) or adjacent cells within the same tissue(planar signals)2–4.

Planar and vertical signals play a crucial role in theinduction and patterning of the rostral neural plate, whichcontains the forebrain, midbrain and rostral-hindbrain primordia. Once early anterior patterning is induced, localorganizer centres that have polarizing and inductive propertiesdevelop within the broadly regionalized neuroectodermin genetically defined positions as transverse rings of neuroepithelium3.

The best-characterized organizer is located at theboundary between the midbrain and the anterior hind-brain (the midbrain–hindbrain boundary, or MHB). Themidbrain corresponds to the mesencephalon, is behind theforebrain and gives rise to structures including the su-perior and inferior colliculi. The anterior hindbrain is behindthe midbrain and develops into the cerebellum and pons,which constitute the metencephalon. Because the organizer isassociated with the isthmic constriction at the MHB, it hasbeen called the isthmic organizer. Planar signals emitted fromthis narrow ring of neuroepithelium have morphogeneticproperties that induce and polarize the midbrain and anteriorhindbrain.

Tissues that can respond to these signals by modifyingtheir fate include the posterior diencephalon, the midbrainand the rhombencephalon. The tissue-responding abilityand inducing properties of the isthmic organizer wereoriginally discovered in transplantation experiments5–7.Increasing amounts of data are now clarifying the mol-ecular nature of the inducing signal(s) and the genetic cas-cade that leads to the establishment, positioning andmaintenance of the isthmic organizer (Fig. 1).

The positioning of the isthmic organizer iscontrolled by Otx2 and Gbx2An organizer is thought to be established gradually withina tissue by a series of steps that first specify different fieldsand then produce morphogenetic properties at theirboundaries8,9. These properties include the expression ofspecific genes, the production of morphogenetic moleculesand the ability to generate cell diversity. Once a boundary

has been defined between two different fields, co-opera-tive cell–cell interactions might eventually produce signalling molecules that activate specific differentiationprogrammes in adjacent cells.

The rostral neural plate is already regionalized by theend of gastrulation, when Otx2 is expressed along the pre-sumptive fore–midbrain region, with a posterior borderthat is adjacent to the anterior border of expression ofGbx2, which, in turn, defines the anterior hindbrain10. Atthe boundary between these two territories, signal mol-ecules such as WNT1 and FGF8 will be expressed in twoadjacent narrow strips (Fig. 1). Previous studies have indi-cated that OTX2 and GBX2 are involved in proper func-tioning of the isthmic organizer and patterning of mid-brain and anterior hindbrain.

Gbx2-null mice10 lack the anterior hindbrain and dis-play abnormal caudal expansion of posterior midbrain,paralleled by posteriorized expression of Otx2, Wnt1 andFgf8. Mutant embryos retaining only one functional alleleof Otx2 (Otx12/2 Otx21/2) show morphological transfor-mation of the caudal diencephalon and mesencephaloninto an enlarged metencephalon, and also coordinate mol-ecular anteriorization of isthmic markers such as GBX2,FGF8, WNT1 and OTX2 itself11,12. Furthermore, embryosin which Otx2 is replaced by Otx1 show no detectableOTX proteins in the neuroectoderm, fail to maintain earlyanterior patterning, are headless and express isthmicmarkers at the rostral tip of the neuroectoderm13.Therefore, these studies indicate that: (1) Gbx2 is requiredfor patterning of the anterior hindbrain and proper estab-lishment of the isthmic organizer10; (2) reducing the levelof OTX proteins below a critical threshold results in ante-rior shift of the MHB11–13; and (3) lack of OTX or GBX2proteins does not prevent the initial expression of eitherFgf8 or any of the other MHB genes tested. These twotranscription factors have also been reported to be crucialfor positioning the isthmic organizer at the MHB of mouseembryos14,15.

Millet et al.14 revisited the early phenotype of Gbx22/2

embryos10 and showed that the first abnormality in geneexpression was a posterior expansion of Otx2 expressionand a posterior shift of Fgf8 and Wnt1 expression10,14.Then, by ectopically expressing a Wnt1-driven Gbx2 trans-gene in the midbrain, they showed that the posterior domainof Otx2 expression became repressed and that the endogen-ous Gbx2, Wnt1 and Fgf8 genes were transcribed in a more-anterior domain (Fig. 2). Thus, these data indicated

Positioning the isthmic organizerwhere Otx2 and Gbx2 meet

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Regional diversity along the anterior–posterior axis of the central nervous system is established duringgastrulation and is subsequently refined by local organizing centres that are located at genetically definedpositions. The isthmic organizer possesses midbrain- and cerebellum-inducing properties, and its positioning atthe midbrain–hindbrain boundary is a crucial event that controls midbrain and cerebellum development. Recentwork has shown that two transcription factors, Otx2 and Gbx2, are instrumental in positioning the isthmicorganizer at the midbrain–hindbrain boundary.

237

Antonio Simeonesimeone@iigbna.iigb.an.cnr.it

International Institute ofGenetics and Biophysics,CNR, Via G. Marconi 12,80125 Naples, Italy; andMRC Centre forDevelopmentalNeurobiology, King’sCollege London, Guy’sCampus, London, UK SE1 9RT.

that GBX2 might repress Otx2 and that GBX2 is requiredto sharpen the caudal border of the Otx2-expressiondomain, which ultimately defines the position of the organizer.

To study the effects of altering the expression domainof Otx2, Broccoli et al. used a knock-in approach involv-ing ectopic expression of an En1-driven Otx2 transgene inmouse rostral hindbrain15. Brains from these mice lacked

most of the medial region of the cerebellum and displayedan enlarged posterior mesencephalon. At early stages ofembryogenesis, as a consequence of the posterior expan-sion of the Otx2 domain, En1, Wnt1 and Fgf8 were alsoshifted to the posterior, whereas Gbx2 and Fgf8 wererepressed in the Otx2 ectopic domain (Fig. 2). These find-ings showed that Otx2 might, by repressing Gbx2, resetthe isthmic organizer to a more-posterior position.Furthermore, the caudal border of Otx2 expression playsa crucial role in this process and correlates withWnt1–Fgf8 expression.

Similar conclusions have been reached by misexpress-ing Otx2 and Gbx2 in chick embryos16. Indeed, this studyindicated that the posterior limit of the midbrain and thesite of Fgf8 expression are determined by interactionbetween Gbx2 and Otx2.

A genetic cascadeThe proper establishment of the isthmic organizer isdefined not just by these two transcription factors butrather by a complex cascade of genetic interactions(Fig. 1). The presumptive fore–midbrain and the prospec-tive rostral hindbrain are visualized at 7.75 days postcoitum (d.p.c) by the expression of Otx2 and Gbx2,respectively (Fig. 1). After this, En1, Wnt1 and Fgf8 areactivated in broad areas along the posterior midbrain andthe rostral hindbrain, and then their expression becomesrestricted. Between 9 and 10 d.p.c., the restricted expres-sion of these genes defines an isthmic molecular code: Fgf8and Gbx2 identify the metencephalic side of the MHB,and Wnt1 and Otx2 identify the mesencephalic side(Fig. 1).

The current view is that Otx2 is needed first in theanterior visceral endoderm to induce early anterior pat-terning and subsequently in the axial mesendodermand/or rostral neural plate (or both) to maintain anteriorpatterning4,17,18. Signals from primitive-streak-derivedmesoderm can induce En1 expression in neuroectoderm;its competence to express En1 depends on a non-cell-autonomous function of Otx219. It has recently beenshown in chicks that En1 activation might be mediated byFgf4 expressed in the notochord rostral to the Hensen’snode and that, once induced, En1 might activate Fgf8expression20,21 (Fig. 1). However, in Zebrafish, Fgf8expression anticipates eng, and Pax2.1 is required to acti-vate eng expression and to maintain Wnt1, eng and Fgf8expression at the MHB22,23.

The analysis of Wnt1 mutants has shown that WNT1is necessary for the maintenance of En1 expression in themidbrain and rostral hindbrain24. Accordingly, in Wnt12/2

mice, the expression of En1 under a Wnt1 promoter frag-ment can rescue Fgf8 expression and the early develop-ment of the midbrain and rostral hindbrain25.Furthermore, the analysis of swaying (a Wnt1 mousemutant with an irregular mesencephalon–metencephalonboundary) has suggested that Wnt1 might also play a role in segregating differently specified mesencephalon–metencephalon cell populations26.

However, the midbrain- and cerebellum-inducing prop-erties of the isthmic organizer have so far only beendemonstrated for FGF827,28. Indeed, when implanted inthe caudal diencephalon, an FGF8-soaked bead caninduce an ectopic midbrain by modifying the fate of thehost tissue surrounding the bead. FGF8 misexpression inthe midbrain also represses Otx2 and induces Gbx2, En1,

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MHB

Mes Met

Wnt1

Otx2

Fgf8

Gbx2

A P

En1

Wnt1Fgf8

Gbx2Otx2

En1

MHB

Mes Met

(a)

(b)

Anterior notochord

Fgf4

FIGURE 1. The genetic cascade that establishesthe isthmic organizer

(a) In mouse embryos, between the late-gastrula and early-somite stage(7.75–8.5 days post coitum), a signal directed from the anterior notochord to theoverlying neuroectoderm activates (1) En1 expression in the mesencephalon(Mes) and metencephalon (Met). At the same time as En1 is expressed, Wnt1 isactivated in the Mes and maintains En1 expression20, which, in turn, might berequired for Fgf8 expression in the anterior Gbx2 domain20,24,25. Evidence in chickembryos20 indicates that: (1) Fgf4 is transiently expressed in the anteriornotochord; (2) Fgf4 might induce En1; and (3) En1 might, in turn, ectopicallyinduce Fgf8. Once induced, the early Fgf8 domain becomes restricted to the Metside of the midbrain–hindbrain boundary (MHB) via a reciprocal negative (redbars) interaction with Otx2 and a positive interaction with Gbx2 14–16,28–30. Anteriorand posterior ends of the notochord are indicated by A and P, respectively. (b) Ata later stage (9.5–10 days post coitum), the Otx2, Wnt1, Gbx2, Fgf8 and En1domains of expression define a molecular code centred on the MHB.Transplantation experiments, Fgf8-soaked-bead implants and animal modelsindicate that the molecular code at the Mes–Met region is maintained by positiveand negative genetic interactions. Positive interactions between Fgf8, En1, Wnt1and Gbx2 maintain their own expression, whereas negative reciprocalinteractions between Otx2 and Gbx2 or Fgf8 maintain a sharp Otx2 posteriorborder at the mesencephalic side of the MHB. Thick lines indicate expressionacross most or all of the neural tube and thin lines represent more-restrictedexpression domains.

Wnt1, Pax5 and Fgf8 itself in adjacent tissue, indicatingthat FGF8-mediated midbrain induction is the conse-quence of a new isthmic-like organizer having been gener-ated in the posterior forebrain27–29. Fgf8-misexpressionstudies also suggest that Gbx2 induction by FGF8 in anOtx21 territory might represent the earliest event thatleads to ectopic formation of an isthmic organizer29.

Together with previous findings, the work of Millet etal.14 and Broccoli et al.15 indicates that: (1) neither Otx2nor Gbx2 are required to initiate Fgf8 expression10,14,17,20,but that Fgf8 could mediate the maintenance of the most-rostral expression of Gbx229; (2) both Gbx2 and Fgf8might repress Otx2, thus sharpening its posterior bor-der14,26,29; and (3) Otx2 might repress both Gbx2 andFgf815,28. Interestingly, two studies have suggested that, inchick embryos, confrontation of the Gbx2-expressing terri-tory with that expressing Otx2 is an important event forthe proper formation of the isthmic organizer30,31. IndeedHidalgo-Sanchez et al.30 showed that an Otx21 graft ofcaudal prosencephalon within the hindbrain induces Gbx2and Fgf8, and represses Otx2 where the grafted tissue con-tacts the host’s Gbx2-expressing territory. Irving andMason31 reported that juxtaposition of the midbrain andrhombomere-1 tissue (but not of the midbrain and anyother rhombomere) is sufficient to induce Fgf8-expressingtissue to gain properties of the isthmic organizer.

Finally, these two papers14,15 also suggest that the levelof the OTX2 and GBX2 proteins is crucial for conferringmesencephalon and metencephalon territorial diversity atthe site of isthmic positioning. Indeed, in Wnt1–Gbx2transgenic embryos, Gbx2 is ectopically expressed alongthe midbrain and, interestingly, the Otx2 caudal limit issharp and located within the most-rostral and weakerdomain of Gbx2, suggesting that there is a minimal thresh-old of Gbx2 for Otx2 repression14. Although this infor-mation is not presented in detail by Broccoli et al.15, themost-posterior domain of Otx2 might slightly invade theGbx2 anterior domain in En1–Otx2 transgenic embryos.This finding might be relevant because the site where theorganizer is reset should result from an equilibrium basedon the local concentrations of the OTX2 and GBX2 pro-teins. It can thus be predicted that mutants with lowerectopic levels of OTX2 and GBX2 should be less efficient atmodifying the position of the isthmic organizer.

The generation of mouse mutants that lack both OTXand GBX proteins, as well as the conditional inactivationof these genes, will greatly contribute to a deeper under-standing of their role in establishing and maintaining theMHB. It is also possible that, during vertebrate evolution,Otx2 and Gbx2 influenced brain morphogenesis bydefining the positioning of the boundary between the

presumptive mesencephalon–metencephalon regions andthereby the extent and differentiation of fore-, mid- andhindbrain territories.

AcknowledgementsI wish to thank D. Acampora and M. Brand for interestingdiscussion on this topic. I am also grateful to A.Secondulfo for typing the manuscript.

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MHB

Mesp1p2ZLI Met

Otx2Gbx2Fgf8Wnt1

Wild type9.5 d.p.c.

Wnt1−Gbx28.5 d.p.c.

En1−Otx29.5 d.p.c.

(b)

(c)

(d)

PA(a)

FIGURE 2. Repositioning the isthmic organizer

(a) The territory between the zona limitans intrathalamica (ZLI) and the posterior border of themetencephalon (Met). This territory includes prosomeres 1 (p1) and 2 (p2), the mesencephalon (Mes)and Met. The anterior (A) and posterior (P) expression domains of key signalling molecules are shownschematically in relation to the tissues of the midbrain–hindbrain boundary (MHB). (b) Expressiondomains of Otx2, Gbx2, Fgf8 and Wnt1 in a wild-type mouse embryo at 9.5 days post coitum (d.p.c.). Theisthmic organizer is positioned at the MHB at the boundary of the Otx2- and Gbx2-expressing domains.(c) Expression domains of these genes in a transgenic mutant mouse embryo (8.5 d.p.c.) that expressesGbx2 under the control of a Wnt1 promoter (Wnt1–Gbx2)14. In Wnt1–Gbx2 mice, the posterior domain ofOtx2 expression is repressed and Gbx2, Wnt1 and Fgf8 are transcribed in a more-anterior domain. (d)Expression domains of these genes in a transgenic mutant mouse embryo (9.5 d.p.c.) that expressesOtx2 under the control of an En1 promoter (En1–Otx2)15. In En1–Otx2 mice, there is a posteriorexpansion of the Otx2-expression domain, and Gbx2 and Fgf8 are repressed in this ectopic Otx2 domain.These data show that the anterior border of Gbx2 and the posterior border of Otx2 are instrumental inpositioning the isthmic organizer.

References1 Vaage, S. (1969) The segmentation of the primitive neural

tube in chick embryos (Gallus domesticus). Adv. Anat.Embryol. Cell Biol. 41, 3–87

2 Ruiz i Altaba, A. (1998) Deconstructing the organizers. Nature391, 748–749

3 Rubenstein, J.L.R. et al. (1998) Regionalization of theprosencephalic neural plate. Annu. Rev. Neurosci. 21,445–477

4 Beddington, R.S. and Robertson, E.J. (1999) Axis developmentand early asymmetry in mammals. Cell 96, 195–209

5 Alvarado-Mallart, R.M. et al. (1990) Pluripotentiality of the 2-day-old avian germinative neuroepithelium. Dev. Biol. 139, 75–88

6 Nakamura, H. (1990) Do CNS anlagen have plasticity indifferentiation? Analysis in quail-chick chimera. Brain Res.511, 122–128

7 Martinez, S. et al. (1991) Induction of mesencephalicphenotype in the 2-day-old chick prosencephalon is precededby the early expression of the homeobox gene en. Neuron 6,971–981

8 Meinhardt, H. (1983) Cell determination boundaries asorganizing regions for secondary embryonic fields. Dev. Biol.96, 375–385

9 Ingham, P.W. and Martinez Arias, A. (1992) Boundaries andfields in early embryos. Cell 68, 221–235

10 Wassarman, K.M. et al. (1997) Specification of the anteriorhindbrain and establishment of a normal mid/hindbrainorganizer is dependent on Gbx2 gene function. Development124, 2923–2934

11 Acampora, D. et al. (1997) Genetic control of brainmorphogenesis through Otx-gene dosage requirement.Development 124, 3639–3650

12 Suda, Y. et al. (1997) Cooperation between Otx1 and Otx2genes in developmental patterning of rostral brain. Mech. Dev.69, 125–141

13 Acampora, D. et al. (1998) Visceral endoderm-restrictedtranslation of Otx1 mediates recovery of Otx2 requirements forspecification of anterior neural plate and normal gastrulation.Development 125, 5091–5104

14 Millet, S. et al. (1999) A role of Gbx2 in repression of Otx2 andpositioning the mid/hindbrain organizer. Nature 401, 161–164

15 Broccoli, V. et al. (1999) The caudal limit of Otx2 expressionpositions the isthmic organizer. Nature 401, 164–168

16 Katakira, Y. et al. (1999) Interaction between Otx2 and Gbx2 definesthe organizing center for the optic tectum. Mech. Dev. 89, 1–10

17 Acampora, D. and Simeone, A. (1999) Understanding the rolesof Otx1 and Otx2 in the control of brain morphogenesis. TrendsNeurosci. 22, 116–122

The mouse t haplotype (see Box 1) occupies a prominentplace in mouse genetics, beginning with its discovery

in 1927 by Dobrovolskaia-Zavadskaia. The t ‘alleles’,which are variants of a ~20 cM region of chromosome 17 called the T/t complex (or simply, t complex), have several remarkable properties. t haplotypes isolated fromaround the world usually contain one of several differentrecessive lethal mutations acting at various developmentalstages. Males carrying two complementing t haplotypesare invariably sterile, and there is recombination suppres-sion in 1/t heterozygotes throughout the t complexregion. Most interesting is that 1/t males transmit the tchromosome to nearly all offspring, the TRD phenomenon.

Genetic analyses of t haplotypes led Mary Lyon to pos-tulate that TRD occurs through the action of several t-haplotype-encoded, trans-acting t complex distorter (Tcd)loci upon the t complex responder (Tcr) locus1. A male heterozygous for Tcr transmits the Tcr-containing chromo-some almost exclusively when all distorters are present. Inthe absence of distorters, the Tcr-containing chromosome istransmitted to about 15% of offspring. The distorters actadditively to increase Tcr transmission (Fig. 1). Thus, as thecentral locus in TRD, identification of Tcr is critical tounderstanding the entire phenomenon. Herrmann et al.

have done just that, using information on the nature of theTcr gene to postulate a mechanism of TRD that invokeskinase-mediated signalling and flagellar development2. Thecloning of Tcr is a landmark in the history of mouse gen-etics, as many of the world’s most prominent mouse geneti-cists and developmental biologists investigated t haplotypesat one time or another.

The diverse phenotypes associated with t haplotypesappear to be intricately linked for the evolutionary purposeof TRD3. Genetic evidence suggests that the genes respon-sible for homozygous male sterility (the t complex sterilityloci, or tcs), are identical to the distorters4. If true, this indi-cates that TRD comes at a price: homozygous sterility. Thisside effect was presumably countered by selective acquisi-tion of recessive lethal mutations to eliminate sterile malesfrom the population; the presence of sterile males wouldhave dire consequences for small groups of mice calleddemes, in which one dominant male mates with a harem offemales5. Shuffling of the TRD genes by recombination isblocked by four chromosomal inversions in t haplotypes,allowing them to be inherited as a unit6. This creates aquagmire for geneticists, preventing high-resolution map-ping of most of the TRD loci; all genetic analyses have used‘partial t haplotypes’, which are rare recombinants thatoccurred across the inversions in 1/t mice.

Segregation distortion of mouse t haplotypesthe molecular basis emerges

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The t haplotype is an ancestral version of proximal mouse chromosome 17 that has evolved mechanisms topersist as an intact genomic variant in mouse populations. t haplotypes contain mutations that affect embryonicdevelopment, male fertility and male transmission ratio distortion (TRD). Collectively, these mutations drive theevolutionary success of t haplotypes, a phenomenon that remains one of the longstanding mysteries of mousegenetics. Molecular genetic analysis of TRD has been confounded by inversions that arose to lock together thevarious elements of this complex trait. Our first molecular glimpse of the TRD mechanism has finally beenrevealed with the cloning of the t complex responder (Tcr) locus, a chimeric kinase with a genetically cisactive effect. Whereas 1 sperm in a 1/t male have impaired flagellar function caused by the deleterious actionof trans-active, t-haplotype-encoded ‘distorters,’ the mutant activity of Tcr counterbalances the distorter effects,maintaining the motility and fertilizing ability of t sperm.

John Schimentijcs@ jax.org

The Jackson Laboratory,Bar Harbor, ME 04609,

USA.

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18 Rhinn, M. et al. (1998) Sequential roles for Otx2 in visceralendoderm and neuroectoderm for forebrain and midbraininduction and specification. Development 125, 845–856

19 Rhinn, M. et al. (1999) Cell-autonomous and non-cell-autonomous functions of Otx2 in patterning the rostral brain.Development 126, 4295–4304

20 Shamim, H. et al. (1999) Sequential roles for Fgf4, En1 andFgf8 in specification and regionalization of the midbrain.Development 126, 945–959

21 Araki, I. and Nakamura, H. (1999) Engrailed defines theposition of dorsal dimesencephalic boundary by repressingdiencephalic fate. Development 126, 5127–5135

22 Reifers, F. et al. (1998) Fgf8 is mutated in zebrafishacerebellar (ace) mutants and is required for maintenance of

midbrain–hindbrain boundary development andsomitogenesis. Development 125, 2381–2395

23 Lun, K. and Brand, M. (1998) A series of no isthmus (noi)alleles of the zebrafish pax2.1 gene reveals multiple signalingevents in development of the midbrain–hindbrain boundary.Development 125, 3049–3062

24 Joyner, A.L. (1996) Engrailed, Wnt and Pax genes regulatemidbrain–hindbrain development. Trends Genet. 12, 15–20

25 Danielian, P.S. and McMahon, A.P. (1996) Engrailed-1 as atarget of the Wnt-1 signalling pathway in vertebrate midbraindevelopment. Nature 383, 332–334

26 Bally-Cuif, L. et al. (1995) Involvement of Wnt-1 in the formationof the mes/metencephalic boundary. Mech. Dev. 53, 23–34

27 Crossley, P.H. et al. (1996) Midbrain development induced by

FGF8 in the chick embryo. Nature 380, 66–6828 Martinez, S. et al. (1999) FGF8 induces formation of an ectopic

isthmic organizer and isthmocerebellar development via arepressive effect on Otx2 expression. Development 126, 1189–1200

29 Liu, A. et al. (1999) FGF8 can activate Gbx2 and transformregions of the rostral mouse brain into a hindbrain fate.Development 126, 4827–4838

30 Hidalgo-Sanchez, M. et al. (1999) Fgf8 and Gbx2 inductionconcomitant with Otx2 repression is correlated withmidbrain–hindbrain fate of caudal prosencephalon.Development 126, 3191–3203

31 Irving, C. and Mason, I. (1999) Regeneration of isthmic tissueis the result of a specific and direct interaction betweenrhombomere 1 and midbrain. Development 126, 3981–3398